Centrifuge tube comprising a floating buoy, and methods for using the same

ABSTRACT

Multi-component separation devices configured to separate components of a liquid sample by centrifugation are provided. Aspects of the separation devices include a container having a distal end and a proximal end and a buoy configured to be displaced along a longitudinal axis within the container where the buoy includes one or more sealed chambers. Also provided are methods of using the subject devices to separate components of a multi-component liquid sample such as whole blood, bone marrow aspirate or stromal vascular fraction as well as systems suitable for practicing the subject methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/508,038, filed Mar. 1, 2017, which is a National Stage Entry ofPCT Application No. PCT/US2015/057599, filed Oct. 27, 2015, which claimspriority from U.S. Provisional Patent Application No. 62/069,783, filedOct. 28, 2014, the disclosures of which are herein incorporated byreference.

INTRODUCTION

Centrifugation has been used in the separation of components in asuspended medium to obtain cells, organelles or macromolecules containedin multi-component biologic fluids including bone marrow, whole blood,peripheral blood, urine, phlegm, synovial semen, milk, saliva, mucus,sputum, exudates, cerebrospinal fluid, amniotic fluid, cord blood,intestinal fluid, cell suspensions, tissue digests, tumor cellcontaining cell suspensions, microbe containing cell suspensions,radiolabelled cell suspensions and cell culture fluid which may or maynot contain additional substances (e.g., anticoagulants to preventclotting). Centrifugation of a medium having suspended particles causesthe particles to sediment in the direction outward from the axis ofrotation. The force generated by centrifugation is proportional to thespeed of rotation and the radius of the rotor. At a fixed force andmedium viscosity, the sedimentation rate of the particle is proportionalto the molecular weight of the particle and the difference between itsdensity and the density of the medium.

Components from biological fluids are used in a variety of therapeutic,diagnostic and research applications. Many biological fluid chemistrytests require separation of the components in the biological fluid. Forexample, when the biological fluid is blood, white blood cells, redblood cells, platelets and plasma components are often separated fortesting. Enriched preparation of biological samples having sufficientconcentration of components for the desired therapeutic, diagnostic orresearch use, can often require numerous and lengthy manipulations whichoften degrades the recovered materials or diminish the amount ofrecoverable biological sample components. For example, multipleiterations of separation and washing of biological samples can bedeleterious to components such as white blood cells, red blood cells andplatelets due to over-processing.

SUMMARY

Multi-component separation devices configured to separate components ofa liquid sample by centrifugation are provided. Aspects of theseparation devices may include a container having a distal end and aproximal end and a buoy configured to be displaced along a longitudinalaxis within the container where the buoy includes one or more sealedchambers containing a vacuum or a fluidic, e.g., gaseous or liquid,composition. Also provided are methods of using the subject devices toseparate components of a multi-component liquid sample, as well assystems suitable for practicing the subject methods.

Aspects of the disclosure include devices for separating components of amulti-component liquid sample by centrifugation. Devices for separatinga multi-component liquid sample (e.g., whole blood or bone marrowaspirate) according to certain embodiments include a container having adistal end and a proximal end and a buoy configured to be displacedalong a longitudinal axis within the container, where the buoy includesone or more sealed chambers containing a vacuum or a fluidic, e.g.,gaseous or liquid, composition. The buoy, in some embodiments, includesa distal frustoconical shaped component and a proximal end having anouter surface, which surface may be a concave or convex outer surface.In certain instances, the buoy includes an orifice at the base of theouter surface at the buoy proximal end and a centrifuge activatedsuspension floor (e.g., in the form of a check valve) having an openposition and a closed position such that the suspension floor isconfigured to fluidically seal the orifice at the base of the outersurface when in the closed position. In other instances, the buoyincludes a first orifice at the base of the outer surface of the buoyproximal end, a second orifice at a position distal to the first orificealong the longitudinal axis of the buoy, a channel that extends from thefirst orifice to the second orifice and a centrifuge activated valve(i.e., check valve) having an open position and a closed position suchthat the valve is configured to fluidically seal the second orifice whenin the closed position. Where the buoy includes a centrifuge activatedvalve, in some instances, the valve is a ball and spring valve, such asa stainless steel ball and spring valve. In certain embodiments, thecontainer includes a cap positioned at the proximal end of the containerhaving one or more ports into the interior cavity of the container. Insome embodiments, the cap positioned at the proximal end of thecontainer consists of a single port. In some instances, the separationdevice also includes a conduit that extends from the port to theproximal end of the buoy, such as at a position along the outer edge ofthe buoy proximal end (i.e., adjacent to the inner walls of thecontainer). The conduit may be releasably attached to or fullyintegrated with one or more of the port and the buoy. In certainembodiments, the conduit is coupled to a stream modulator at the buoyproximal end. The stream modulator may also be attached to the buoy.Predetermined volume measurement markings may also, in certaininstances, be present on the outer walls of the container.

Aspects of the disclosure also include methods for separating componentsof a multi-component liquid sample. Methods according to certainembodiments include introducing a multi-component liquid sample (e.g.,blood) into a container of a separation device having a buoy configuredto be displaced along a longitudinal axis within the container where thebuoy includes one or more sealed chambers containing a fluidic, e.g.,gaseous or liquid, composition, subjecting the sample to a force ofcentrifugation to produce two or more fractions in the sample, eachfraction having a component from the sample of a different density andcollecting one or more components of the sample. Where the containerincludes a cap at the proximal end having only a single port, the sampleis introduced into the container and one or more fractions may becollected from the container after centrifugation through the singleport. For example, where the single port is coupled to a conduit, thesample may be introduced into the container through the port and conduitand one or more fractions from the multicomponent sample may becollected after centrifugation through the conduit and single port. Insome embodiments, the container includes a cap at the proximal endhaving more than one port. In some instances, the container includes asecond opening in the cap to allow air to vent during sampleintroduction and removal.

In certain embodiments, the sample is a biological sample (e.g., bloodor bone marrow aspirate) and methods include introducing a biologicalsample into the container of a separation device having a buoyconfigured to be displaced along a longitudinal axis within thecontainer where the buoy includes one or more sealed chambers containinga fluidic composition, subjecting the blood sample to a force ofcentrifugation to produce two or more fractions in the biologicalsample, each fraction having a biological component of a differentdensity and collecting one or more of the separated components. In someinstances, subjecting the biological sample to a force of centrifugationis sufficient to displace the buoy proximally along a longitudinal axiswithin the container from the bottom of the container to a position atthe interface between a first fraction and a second fraction of thebiological sample. In certain embodiments, collecting one or morecomponents of the biological sample includes removing a portion of afirst separated fraction of the biological sample, mixing the remainingportion of the first separated fraction with a second separated fractionwithin the buoy to produce a mixture of the first separated fraction andthe second separated fraction and removing the mixture from thecontainer.

In other embodiments, the present invention provides a means ofcollecting varying volumes of one or more components of the sample by astep comprising positioning the container of the separation device at afirst angle (e.g., 20 degrees or more) with respect to an axisorthogonal to the ground, removing a portion of a first separatedfraction of the sample through a conduit which extends from a port inthe cap to the proximal end of the buoy, rotating the container by asecond angle (e.g., 180 degrees) along the longitudinal axis of thecontainer, aspirating the remaining portion of the first separatedfraction of the sample through the conduit, mixing the remaining portionof the first separated fraction with a second separated fraction withinthe buoy to produce a mixture of the first separated fraction and thesecond separated fraction and removing the mixture from the container.In some instances, the first fraction contains platelet poor plasma andthe second fraction contains white blood cells and platelet rich plasma.

In still other embodiments, the present invention provides means ofcollecting varying volumes of one or more components of the sample by astep that includes positioning the device at a first angle with respectto an axis orthogonal to the ground (e.g., in a tilter stand); removinga portion of a first fraction of the biological sample; tilting thedevice to a second angular position with respect to the axis orthogonalto the ground; aspirating the remaining portion of the first fraction ofthe biological sample; mixing the remaining portion of the firstfraction with a second fraction within the buoy to produce a mixture ofthe first fraction and the second fraction; and removing the mixturefrom the container. In some instances, the first fraction containsplatelet poor plasma and the second fraction contains white blood cellsand platelet rich plasma.

Aspects of the disclosure also include systems for practicing thesubject methods. Systems according to certain embodiments include acentrifuge and one or more of the subject separation devices thatinclude a container having a distal end and a proximal end and a buoyconfigured to be displaced along a longitudinal axis within thecontainer where the buoy includes one or more sealed chambers containinga fluidic, e.g., gaseous or liquid, composition.

BRIEF DESCRIPTION OF THE FIGURES

The invention may be best understood from the following detaileddescription when read in conjunction with the accompanying drawings.Included in the drawings are the following figures:

FIG. 1 depicts a side-view of a separation device having a buoypositioned inside of a container having a ball-and-spring valveaccording to certain embodiments.

FIG. 2 depicts a side-view of a separation device having a buoypositioned inside of a container having a ball-and-spring valveaccording to certain embodiments.

FIG. 3A illustrates a three-dimensional view of an example of a devicefor separating components of a multi-component liquid according tocertain embodiments.

FIG. 3B illustrates a side view of an example of a device for separatingcomponents of a multi-component liquid according to certain embodiments.

FIG. 3C illustrates an example of a device having a multi-componentliquid separated into two or more regions (i.e., fractions) according tocertain embodiments.

FIG. 3D illustrates an example of a device having a multi-componentliquid separated into two or more regions (i.e., fractions) according tocertain embodiments.

FIG. 4A depicts a three-dimensional view of a device for separatingcomponents of a multi-component liquid according to certain embodiments.

FIG. 4B depicts a three-dimensional view of a device for separatingcomponents of a multi-component liquid according to certain embodiments.

FIG. 4C depicts a side view of a device for separating components of amulti-component liquid according to certain embodiments.

FIG. 4D depicts a different side view of a device for separatingcomponents of a multi-component liquid according to certain embodiments.

FIG. 5A illustrates a first step of a step-by-step method of FIGS. 5A-5Ffor separating components of a multi-component liquid sample (e.g.,blood or bone marrow aspirate) according to certain embodiments. Aseparation device having multi-component sample in the container beforeapplying a force of centrifugation is depicted.

FIG. 5B illustrates a second step of a step-by-step method of FIGS.5A-5F for separating components of a multi-component liquid sample(e.g., blood or bone marrow aspirate) according to certain embodiments.The subject device is depicted with introduced sample at the beginningof centrifugation where the ball and spring valve in the buoy is in aclosed position.

FIG. 5C illustrates a third step of a step-by-step method for separatingcomponents of a multi-component liquid sample (e.g., blood or bonemarrow aspirate) according to certain embodiments. The device isdepicted with the ball and spring valve in the open position duringcentrifugation due to the force of the ball compressing the spring.

FIG. 5D illustrates a fourth step of a step-by-step method forseparating components of a multi-component liquid sample (e.g., blood orbone marrow aspirate) according to certain embodiments. The device isdepicted with the components of the sample separated at differentpositions in the device.

FIG. 5E illustrates a step-by-step method for separating components of amulti-component liquid sample (e.g., blood or bone marrow aspirate)according to certain embodiments. The device is depicted aftercollection of separated components of the sample according to certainembodiments.

FIG. 5F illustrates step-by-step methods for separating components of amulti-component liquid sample (e.g., blood or bone marrow aspirate)according to certain embodiments. The device is depicted aftercollection of separated components of the sample according to certainembodiments.

FIG. 6A illustrates a step of removing in an example of methods forseparating components of a multi-component liquid sample (e.g., blood orbone marrow aspirate) according to certain embodiments.

FIG. 6B illustrates a step of removing in an example of methods forseparating components of a multi-component liquid sample (e.g., blood orbone marrow aspirate) according to certain embodiments.

FIG. 7A illustrates step-by-step methods for separating components of amulti-component liquid sample according to certain embodiments. Amulti-component liquid sample (e.g., blood) is introduced into thecontainer with a syringe or other suitable dispensing protocol.

FIG. 7B illustrates step-by-step methods for separating components of amulti-component liquid sample according to certain embodiments. Themulti-component sample is separated into fractions.

FIG. 7C illustrates step-by-step methods for separating components of amulti-component liquid sample according to certain embodiments. Thedevice is first placed into a receptacle of a tilter stand.

FIG. 7D illustrates step-by-step methods for separating components of amulti-component liquid sample according to certain embodiments. Thereceptacle is pivoted to a first angular position.

FIG. 7E illustrates step-by-step methods for separating components of amulti-component liquid sample according to certain embodiments. Thedevice comprising the remaining portion of the first fraction after aportion of a first fraction of the sample is aspirated.

FIG. 7F illustrates step-by-step methods for separating components of amulti-component liquid sample according to certain embodiments. Thereceptacle is pivoted to a second angular position.

FIG. 7G illustrates step-by-step methods for separating components of amulti-component liquid sample according to certain embodiments. Theremaining portion of the first fraction is aspirated and reintroducedback into the device to mix with a second fraction.

FIG. 7H illustrates step-by-step methods for separating components of amulti-component liquid sample according to certain embodiments. Themixture is removed from the container.

FIG. 8 depicts an example flow diagram of separating components of abiological sample according to certain embodiments.

FIG. 9A illustrates an example of a support for positioning one or moreof the subject devices described above at an angle according to certainembodiments. The device is positioned in the support with the distal endin the support cradle with access to the inlet/outlet port.

FIG. 9B illustrates an example of a support for positioning one or moreof the subject devices described above at an angle according to certainembodiments.

FIG. 10A illustrates an example of placing one or more of the subjectdevices at an angle in a support to collect one or more components of aseparated multi-component liquid according to certain embodiments. Thedevice is positioned at an angle with the distal portion of thecontainer inserted into the support with access to inlet/outlet port. Inthis embodiment, the angular position indicator on the side of thedevice is lined up with a mark on the support.

FIG. 10B illustrates an example of placing one or more of the subjectdevices at an angle in a support to collect one or more components of aseparated multi-component liquid according to certain embodiments. Anexploded view of the device where the buoy port is at the lowestpossible position (e.g., the concave outer surface of the buoy proximalend slopes downward) which facilitates removal of the separatedcomponents of the multi-component liquid.

FIG. 11A depicts an adjustable tilter stand for positioning the deviceat a desired angle to remove one (or a portion) or more fractions aftercentrifugation according to certain embodiments. A three-dimensionalperspective of the tilter stand.

FIG. 11B depicts an adjustable tilter stand for positioning the deviceat a desired angle to remove one (or a portion) or more fractions aftercentrifugation according to certain embodiments. A three-dimensionalperspective of the tilter stand.

FIG. 11C depicts an adjustable tilter stand for positioning the deviceat a desired angle to remove one (or a portion) or more fractions aftercentrifugation according to certain embodiments. The tilter stand from afront-facing perspective.

DETAILED DESCRIPTION

Multi-component separation devices configured to separate components ofa liquid sample by centrifugation are provided. Aspects of theseparation devices may include a container having a distal end and aproximal end and a buoy configured to be displaced along a longitudinalaxis within the container where the buoy includes one or more sealedchambers containing a fluidic, e.g., gaseous or liquid, composition.Also provided are methods of using the subject devices to separatecomponents of a multi-component liquid sample, as well as systemssuitable for practicing the subject methods.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

As summarized above, the present disclosure provides a separation devicefor separating components of a liquid sample. In further describingembodiments of the disclosure, separation devices that include acontainer having a distal end and a proximal end and a buoy configuredto be displaced along a longitudinal axis within the container are firstdescribed in greater detail. Next, methods for separating components ofa liquid sample, such as a blood sample, with the subject separationdevices are described. Systems, including a centrifuge, suitable forpracticing the subject methods are also provided.

Devices for Separating Components of a Liquid Sample by Centrifugation

As summarized above, aspects of the present disclosure include devicesfor separating components of a multi-component liquid sample bysubjecting the sample to a force of centrifugation. The term“separating” is used herein in its conventional sense to refer to thephysical separation of a plurality of components based on a particularphysical or chemical property, such as density of the component. Asdescribed in greater detail below, the multi-component sample isintroduced into one or more of the subject separation devices andsubjected to a force of centrifugation for a duration sufficient toseparate one or more components of the liquid sample. In embodiments,components are separated within the sample such that each component hasan increased concentration in a particular region (e.g., distal end,proximal end or middle portion of the device container) as compared tothe multi-component sample before centrifugation. In certainembodiments, the multi-component liquid sample is blood or a derivativethereof and separation devices of interest are configured to separatecomponents of the blood sample, such as separating white blood cells,red blood cells, plasma and platelets.

In embodiments, components of the liquid sample are separated into twoor more regions (i.e., fractions) in the sample such that 5% or more ofa certain component is separated into a particular region (e.g., distalend, proximal end or middle portion) of the device container, such as10% or more, such as 20% or more, such as 25% or more, such as 30% ormore, such as 40% or more, such as 50% or more, such as 60% or more,such as 70% or more, such as 80% or more, such as 90% or more, such as95% or more and including separating 99% or more of a component into aparticular region of the device container. In certain embodiments, 100%of the component is separated into a particular region of the devicecontainer. For example, where the multi-component liquid sample is ablood sample, separation devices of interest are configured to separatethe blood sample in two or more fraction layers, such as three or morefraction layers in the device container. For example, in certainembodiments a layer of red blood cells forms at a distal end of thedevice container, a layer of platelet poor plasma is formed at theproximal end of the device container and a layer of buffy coat is formedon a surface of the buoy.

As used herein, the term “multi-component liquid sample” is used todescribe suspended media having more than one component, wheremulti-component liquid samples may include, but are not limited to,biological samples. The term “biological sample” is used in itsconventional sense to include a whole organism, plant, fungi or a subsetof animal tissues, cells or component parts which may in certaininstances be found in blood (e.g., peripheral blood), mucus, lymphaticfluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolarlavage, amniotic fluid, amniotic cord blood, urine, vaginal fluid andsemen. As such, a “biological sample” refers to both the native organismor a subset of its tissues as well as to a homogenate, lysate or extractprepared from the organism or a subset of its tissues, including but notlimited to, for example, plasma, serum, spinal fluid, lymph fluid,sections of the skin, respiratory, gastrointestinal, cardiovascular, andgenitourinary tracts, tears, saliva, milk, blood cells, tumors, organs.Biological samples may include any type of organismic material,including both healthy and diseased components (e.g., cancerous,malignant, necrotic, etc.). Biological samples may include biologicfluids including bone marrow, phlegm, sputum, exudates, intestinalfluid, cell suspensions, tissue digests, tumor cell containing cellsuspensions, microbe containing cell suspensions, and radiolabelled cellsuspensions.

In certain embodiments, the biological sample is a liquid sample, suchas whole blood or derivative thereof (e.g., plasma), tears, sweat,urine, semen, etc., where in some instances the sample is a bloodsample, including whole blood, such as blood obtained from venipunctureor fingerstick (where the blood may or may not be combined with anyreagents prior to assay, such as preservatives, anticoagulants, etc.).The term “blood sample” refers to whole blood or a subset of bloodcomponents, including but not limited to platelets, red blood cells,white cells, buffy coat and blood plasma. The term “buffy coat” is usedherein in its conventional sense to refer to the fractionated portion ofblood of intermediate density (less dense than red blood cells, moredense than plasma) that contains white blood cells and platelets. Insome embodiments, the blood sample is obtained from an in vivo sourceand can include blood samples obtained from tissues (e.g., bone marrowaspirate, cell suspension from a tissue biopsy, cell suspension from atissue sample, etc.) or directly from a subject. In some cases, bloodsamples derived from a subject are cultured, stored, or manipulatedprior to evaluation.

In certain embodiments the source of the biological sample is a “mammal”or “mammalian”, where these terms are used broadly to describe organismswhich are within the class mammalia, including the orders carnivore(e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), andprimates (e.g., humans, chimpanzees, and monkeys). In some instances,the subjects are humans. The methods may be applied to samples obtainedfrom human subjects of both genders and at any stage of development(i.e., neonates, infant, juvenile, adolescent, adult), where in certainembodiments the human subject is a juvenile, adolescent or adult. Whilethe present disclosure may be applied to samples from a human subject,it is to be understood that the methods may also be carried-out onsamples from other animal subjects (that is, in “non-human subjects”)such as, but not limited to, birds, mice, rats, dogs, cats, livestockand horses.

Separation devices of interest may be configured to separate componentsof multi-component liquid samples of varying size, depending on the sizeof the container and buoy (as described in greater detail below) wherein some instances the volume of sample may range from 5 mL to 5000 mL,such as from 10 mL to 2500 mL, such as from 15 mL to 1000 mL, such asfrom 25 mL to 750 mL, such as from 30 mL to 500 mL, such as from 40 mLto 250 mL, and including from 50 mL to 100 mL. In one example,separation devices of interest are configured to separate components ofa 30 mL sample. In another example, separation devices of interest areconfigured to separate components of a 60 mL sample. In yet anotherexample, separation devices of interest are configured to separatecomponents of a 100 mL sample.

In certain embodiments, the liquid sample is a specimen (e.g., blood orbone marrow aspirate) that has been preloaded into the separation devicecontainer and is stored in the container for a predetermined period oftime before subjecting the sample to centrifugation. For example, asample may be preloaded into one or more of the subject separationdevices and stored at reduced temperature (e.g., refrigerator orfreezer). The amount of storage time before subjecting the sample to aforce of centrifugation may vary, such as 0.1 hours or more, such as 0.5hours or more, such as 1 hour or more, such as 2 hours or more, such as4 hours or more, such as 8 hours or more, such as 16 hours or more, suchas 24 hours or more, such as 48 hours or more, such as 72 hours or more,such as 96 hours or more, such as 120 hours or more, such as 144 hoursor more, such as 168 hours or more and including preloading the sampleinto one or more of the subject separation devices 240 hours or morebefore subjecting the sample to a force of centrifugation or may rangesuch as from 0.1 hours to 240 hours before subjecting the sample to aforce of centrifugation, such as from 0.5 hours to 216 hours, such asfrom 1 hour to 192 hours and including from 5 hours to 168 hours.

In some embodiments, the sample may be preloaded into one or more of thesubject separation devices at a remote location (e.g., at home using anat-home kit or in a physician's office) and sent to a laboratory forprocessing in accordance with the subject methods. By “remote location”is meant a location other than the location at which the sample isobtained and preloaded into the container. For example, a remotelocation could be another location (e.g., office, lab, etc.) in the samecity, another location in a different city, another location in adifferent state, another location in a different country, etc., relativeto the location of the separation device, e.g., as described in greaterdetail below. In some instances, two locations are remote from oneanother if they are separated from each other by a distance of 10 m ormore, such as 50 m or more, including 100 m or more, e.g., 500 m ormore, 1000 m or more, 10,000 m or more, etc.

As summarized above, the subject devices include a buoy having one ormore sealed chambers, such as sealed chambers containing a vacuum or afluidic, e.g., gaseous or liquid, composition. The term “buoy” is usedherein in its conventional sense to refer to an internal movablecomponent or assembly of components of specific aggregate density thatare configured to be displaced along a longitudinal axis within thecontainer during centrifugation. The term “displace” refers to movementof the buoy through the sample in the container during centrifugation.In some embodiments, the buoy is configured to be displaced through thesample in response to the force of centrifugation. In embodiments, thesubject buoy is configured to be displaced along the longitudinal axiswithin the container and can be displaced along all or part of thelength of the inner cavity of the container, such as 25% or more of thelength of the container, such as 35% or more, such as 50% or more, suchas 60% or more, such as 75% or more, such as 90% or more, such as 95% ormore, such as 97% or more and including 99% or more of the length of thecontainer. In certain embodiments, the buoy can be displaced along theentire (i.e., 100%) length of the container.

In some embodiments, the buoy has a density which is greater than themulti-component liquid sample (e.g., whole blood or bone marrowaspirate) and prior to subjecting the sample to a force ofcentrifugation (as described in greater detail below), the buoy ispositioned at the distal end of the tube (i.e., at the bottom when thetube is positioned vertically on a surface parallel to the axis of theground). During centrifugation, the buoy is configured to be displacedproximally along the longitudinal axis of the container and takes afinal position, depending on the type of multicomponent liquidcomposition (as described in greater detail below) that is on top of,below, or within the sample after centrifugation is complete. In someinstances, the buoy is configured to take a final position at theinterface between a first separated fraction and a second separatedfraction of the sample. In other instances, the buoy is configured totake a final position within a fraction of the sample. In yet otherinstances, the buoy is configured to take a final position that is ontop of the fractionated sample. In still other instances, the buoy isconfigured to take a final position that is below the fractionatedsample (e.g., at the bottom of the container). For example, where theliquid sample is whole blood, the buoy may be configured to take a finalposition at an interface between the fraction containing red blood cellsand the fraction containing plasma. In other instances, the buoy isconfigured to take a final position within the fraction containing redblood cells. In other instances, the buoy is configured to take a finalposition wherein the platelets, lymphocytes, monocytes and stem cellscan be extracted without extracting a substantial percentage of thegranulocytes and red blood cells.

In embodiments, the buoy includes one or more sealed chambers. The term“sealed” is used herein in this conventional sense to mean that thechambers are closed from fluidic, e.g., gaseous and liquid,communication with the outside environment of the buoy. Depending on theoverall density of buoy desired, buoys of interest may include one ormore sealed chambers, such as 2 or more, such as 3 or more, such as 4 ormore, such as 5 or more, such as 10 or more and including 25 or moresealed chambers containing a fluidic composition. Where the subjectbuoys include more than one sealed chamber, the chambers may have thesame size, different size, same shape, different sample or anycombination thereof. Each sealed chamber may have a volume which varies,ranging from 0.01 cm³ to 10 cm³, such as from 0.05 cm³ to 9.5 cm³, suchas from 0.1 cm³ to 9 cm³, such as from 0.5 cm³ to 8.5 cm³, such as from1 cm³ to 8 cm³, such as from 1.5 cm³ to 7.5 cm³, such as from 2 cm³ to 7cm³, and including from 2.5 cm³ to 5 cm³. Depending on the number ofsealed chambers present in the subject buoys, the cumulative volumeoccupied by the sealed chambers may range from 0.01 cm³ to 100 cm³, suchas from 0.05 cm³ to 75 cm³, such as from 0.1 cm³ to 50 cm³, such as from0.5 cm³ to 25 cm³, and including from 1 cm³ to 10 cm³. In embodiments,the one or more sealed chambers occupies 25% or more of the total volumeof the buoy, such as 30% or more, such as 35% or more, such as 40% ormore, such as 45% or more, such as 50% or more, such as 60% or more,such as 70% or more and including 75% or more of the total volume of thebuoy.

In some embodiments of the present disclosure, the sealed chamber(s) inthe subject buoys contain a fluidic composition, e.g., a gaseouscomposition, a liquid composition or a combination thereof. In otherembodiments, the sealed chamber(s) has a vacuum within one or more ofthe sealed chambers.

In some embodiments, the subject buoys include one or more sealedchambers having a gaseous composition. In other embodiments, the subjectbuoys include one or more sealed chambers having a liquid composition.In other embodiments, the subject buoys include one or more sealedchambers that contain a vacuum. In yet other embodiments, the subjectbuoys include one or more sealed chambers having a gaseous compositionand one or more sealed chambers having a liquid composition. In stillother embodiments, the subject buoys include one or more sealed chambershaving a gaseous composition, and one or more sealed chambers thatcontain a vacuum. In still other embodiments, the subject buoys includeone or more sealed chambers having a liquid composition and one or moresealed chambers that contain a vacuum. In still other embodiments, thesubject buoys include one or more sealed chambers having a gaseouscomposition, one or more sealed chambers having a liquid composition andone or more sealed chambers that contain a vacuum.

Examples of gases that may be present in the gaseous compositionsinclude, but are not limited to air, carbon dioxide, oxygen, nitrogen,hydrogen, helium, argon, xenon or a combination thereof. In certainembodiments, the sealed chambers contain air. Where the subject buoysinclude more than one sealed chamber, each chamber may contain the sameor a different gaseous composition. For example, each sealed chamber maycontain 1 or more types of gases, such as 2 or more types of gases, suchas 3 or more types of gases and including 5 or more types of gases. Theamount of gas present in the sealed chambers may vary depending on thetype of the gas present and the desired density of the buoy. In someembodiments, the sealed chamber may include 0.001 mmoles or more of thegaseous composition, such as 0.005 mmoles or more, such as 0.01 mmolesor more, such as 0.05 mmoles or more, such as 0.1 mmoles or more, suchas 0.5 mmoles or more and including 0.75 mmoles or more of the gaseouscomposition. The gaseous composition in the sealed chamber may be underpositive or negative pressure, as desired, with respect to atmosphericpressure. In some instances, the pressure of the gaseous composition inthe sealed chamber is less than atmospheric pressure, such as a pressureof 750 torr or less, such as 500 torr or less, such as 400 torr or less,such as 300 torr or less, such as 200 torr or less, such as 100 torr orless, such as 50 torr or less, such as 10 torr or less, such as 1 torror less, such as 0.1 torr or less, such as 0.01 torr or less andincluding where the gaseous composition in the sealed chamber is presentat a pressure of 0.001 torr or less. In other instances, the pressure ofthe gaseous composition in the sealed chamber is greater thanatmospheric pressure, such as a pressure of 775 torr or more, such as1000 torr or more, such as 1500 torr or more, such as 2000 torr or more,such as 2500 torr or more, such as 3000 torr or more, such as 3500 torror more and including where the gaseous composition in the sealedchamber is present at a pressure of 5000 torr or more. In certaininstances, the gaseous composition is present in the sealed chamber atatmospheric pressure (i.e., 760 torr).

In other embodiments, the sealed chamber(s) in the subject buoys containa liquid composition. Depending on the desired density of the buoy,liquid compositions present in the sealed chamber may vary and mayinclude both aqueous and non-aqueous liquid compositions. Accordingly,the density of the liquid composition may be varied as desired (such asby mixing two or more liquid compositions) and may range from 0.5 g/mLto 2 g/mL, such as from 0.6 g/mL to 1.9 g/mL, such as from 0.7 g/mL to1.8 g/mL, such as from 0.8 g/mL to 1.7 g/mL, such as from 0.9 g/mL to1.6 g/mL and including from 1 g/mL to 1.5 g/mL. In some instances, theliquid composition is an aqueous composition, such as aqueous buffers,including but not limited to phosphate buffers (e.g., PBS),tris-buffers, citrate buffers (e.g., sodium citrate), acetate buffers(e.g., sodium acetate) borate buffers (e.g., borax) as well as othertypes of salt buffers such as aqueous buffers containing one or more ofsodium citrate, sodium acetate, sodium phosphate, sodium tartrate,sodium succinate, sodium maleate, magnesium acetate, magnesium citrate,magnesium phosphate, ammonium acetate, ammonium citrate, ammoniumphosphate, and combinations thereof. In other instances, the liquidcomposition is a non-aqueous composition, such as an alcohol, includingbut not limited to methanol, ethanol, propanol, isopropanol, butanol,isobutanol, pentanol, isopentanol, hexanol, heptanol, octanol, nonanol,decanol, undecanol, isoamyl alcohol, benzyl alcohol, cetyl alcohol.Other suitable non-aqueous compositions may include but are not limitedto organic solvents, such as ether, tetrahydrofuran, acetonitrile,dimethyl sulfoxide, ethyl acetate, methylene chloride, chloroform,liquid aliphatic alkanes such as pentane, cyclopentane, hexanes,heptane, iso-octane, xylenes, benzene, toluene, petroleum ether, methylisobutyl ketone, methyl ethyl ketone, pyridine, dioxane, anddimethylformamide, among other organic solvents. In certain instances,the sealed chamber includes a polyhydric alcohol, including but notlimited to glycerol, propylene glycol, neopentyl glycol, diethyleneglycol, pentaerythritol, dipentaerythritol, ethylene glycol,trimethylolpropane, trimethylol ethane, di-trimethylol propane,1,6-hexane diol and combinations thereof. Where the subject buoysinclude more than one sealed chamber, each chamber may contain the sameor different liquid compositions. For example, each sealed chamber maycontain 1 or more types of liquid composition, such as 2 or more typesof liquid compositions, such as 3 or more types of liquid compositionsand including 5 or more types of liquid compositions.

The shape of the sealed chamber(s) may vary, where cross-sectionalshapes of interest include, but are not limited to rectilinearcross-sectional shapes, e.g., squares, rectangles, trapezoids,triangles, hexagons, etc., curvilinear cross-sectional shapes, e.g.,circles, ovals, as well as irregular shapes, e.g., a parabolic bottomportion coupled to a planar top portion. In some embodiments, the sealedchambers are cylindrically shaped. In other embodiments, the sealedchambers are spherical. In yet other embodiments, the sealed chambersare square-shaped. In still other embodiments, the sealed chambers arerectangular.

The sealed chamber(s) may be at any convenient position within the buoy.In some embodiments, the sealed chambers are positioned within the buoyin a random pattern. In other embodiments, the sealed chambers arepositioned in a non-random pattern (i.e., in a predetermined pattern),including in a line pattern or in a pattern of a specific shape (e.g.,taking the same shape as the buoy, as a circle, triangle, etc.). In oneexample, the sealed chambers are arranged along the perimeter within thebuoy. In another example, a buoy may include 2 sealed chamberspositioned on opposite sides of the buoy. In another example, the buoyincludes 4 sealed chambers positioned in a square pattern.

As described above, buoys of interest are configured to be displacedalong a longitudinal axis within the container. Buoys may be anysuitable shape so long as they are capable of being displaced along thelongitudinal axis within the container. In some embodiments, the buoy isfrustoconical-shaped. In other embodiments, the buoy includes acylindrical-shaped proximal portion and a frustoconical-shaped distalportion. In embodiments, the distal end may have a planar, convex orconcave outer surface. In some instances, the distal end has a convexouter surface. For example, the buoy may have a frustoconical-shaped orcylindrical shaped distal end having a convex outer surface. In otherinstances, the distal end has a concave outer surface. For example, thebuoy may have a frustoconical-shaped or cylindrical shaped distal endhaving a concave outer surface.

The outer surface of the proximal end of the buoy may vary, as desired.As such, the proximal end may be planar, concave or convex. In certainembodiments, the buoy includes a proximal end that has a concave outersurface. In these embodiments, the concave outer surface may beconfigured to collect one or more components of the sample and may havea volume which ranges from 0.5 cm³ to 100 cm³, such as from 1 cm³ to 75cm³, such as from 2 cm³ to 50 cm³, such as from 3 cm³ to 25 cm³, andincluding from 5 cm³ to 10 cm³. In certain embodiments, the buoyincludes a proximal end that has a convex outer surface.

In embodiments, the buoy has an orifice at the base of the outersurface. In certain instances, the outer surface at the proximal end ofthe buoy terminates in a flat surface that includes one or moreorifices. For example the base of the concave outer surface at the buoyproximal end may include 2 or more orifices, such as 3 or more, such as4 or more, such as 5 or more and including 10 or more orifices. In otherinstances, the outer surface at the buoy proximal end terminates at anorifice. The orifice may have any suitable cross-sectional shape whereexamples of cross-sectional shapes include, but are not limited torectilinear cross-sectional shapes, e.g., squares, rectangles,trapezoids, triangles, hexagons, etc., curvilinear cross-sectionalshapes, e.g., circles, ovals, etc., as well as irregular shapes, e.g., aparabolic bottom portion coupled to a planar top portion, etc. Dependingon the size of the buoy, amount of liquid sample and specific componentsbeing separated, the size of each orifice may vary, for example rangingfrom 1 mm to 50 mm, such as from 2 mm to 40 mm, such as from 3 mm to 30mm and including from 5 mm to 25 mm.

In some embodiments, the buoy also includes a centrifuge activated valve(which may also be referred to as a check valve) having an open positionand a closed position such that the valve is configured to fluidicallyseal the orifice when in the closed position. By “fluidically seal” ismeant that when the valve is in the closed position, components of theliquid sample, including fluidic components are substantially incapableof passing through the orifice. In other words, little to no amount ofthe liquid sample passes through the orifice when the centrifugeactivated valve is in the closed position. For example, 10% or less ofthe liquid sample passes through the orifice when the centrifugeactivated valve is in the closed position, such as 5% or less, such as3% or less, such as 2% or less, such as 1% or less, such as 0.5% or lessand including 0.1% or less of the liquid sample passes through theorifice when the centrifuge activated valve is in the closed position.

The term “centrifuge activated” is used herein in its conventional senseto refer to opening of the valve (i.e., release the fluidic seal at theorifice) in response to a force of centrifugation. As described ingreater detail below, the term “force of centrifugation” refers to theforce applied to the sample through revolving the subject devices aboutan axis of rotation where the force on the components of the sample isin certain embodiments, given by the relative centrifugal force (RCF).As such, the buoy includes a valve that fluidically seals the orificewhen in the closed position and is opened in response to the appliedforce of centrifugation. In embodiments, the centrifuge activated valvemay be configured to open in response to a force of centrifugation thatvaries, and may range from 1 g to 50,000 g, such as from 2 g to 45,000g, such as from 3 g to 40,000 g, such as from 5 g to 35,000 g, such asfrom 10 g to 25,000 g, such as from 100 g to 20,000 g, such as from 500g to 15,000 g, and including from 1000 g to 10,000 g.

Any convenient centrifuge activated valve protocol may be employed tofluidically seal the orifice in the subject buoys. In certainembodiments, the centrifuge activated valve includes a spring, such aswhere the valve includes a suspension floor which fluidically seals theorifice and is coupled to a spring that expands or compresses inresponse to the force of centrifugation. For example, the centrifugeactivated valve, in certain embodiments, includes a spring that has acompression spring rate that is 0.0001 N/mm or more, such as 0.0005 N/mmor more, such as 0.001 N/mm or more, such as 0.005 N/mm or more, such as0.01 N/mm or more, such as 0.05 N/mm or more, such as 0.1 N/mm or more,such as 0.5 N/mm or more, such as 1 N/mm or more, such as 5 N/mm ormore, such as 10 N/mm or more, such as 25 N/mm or more, such as 50 N/mmor more, such as 100 N/mm or more, such as 250 N/mm or more andincluding 500 N/mm or more.

In certain embodiments, the centrifuge activated valve includes acentrifuge activated suspension floor. In these embodiments, thesuspension floor is configured to open and close in response to theforce of centrifugation. For example, the suspension floor may becoupled to a spring which compresses or expands in response to the forceof centrifugation, as described above. Depending on the type of orificein the buoys, centrifuge activated suspension floors may have anysuitable mass having a cross-sectional shape to form a fluidic seal inthe closed position. Examples of shapes for centrifuge-activatedsuspension floors of interest include, but are not limited torectilinear cross-sectional shapes, e.g., squares, rectangles,trapezoids, triangles, hexagons, etc., curvilinear cross-sectionalshapes, e.g., circles, ovals, etc., as well as irregular shapes, e.g., aparabolic bottom portion coupled to a planar top portion, etc. Incertain embodiments, the centrifuge activated suspension floor is athree-dimensional shape, such as a ball. Depending on the size of theorifice, the centrifuge activated suspension floor may have a width thatis 1 mm or larger, such as 2 mm or larger, such as 5 mm or larger, suchas 10 mm or larger, such as 25 mm or larger and including 50 mm orlarger. For example, the centrifuge activated suspension floor may havea width that ranges from 1 mm to 50 mm, such as from 2 mm to 40 mm, suchas from 3 mm to 30 mm and including from 5 mm to 25 mm.

The centrifuge activated suspension floor may be formed from glass,metal or plastic, such as a flexible or rigid plastic, polymeric orthermoplastic materials. For example, suitable polymeric plastics mayinclude polycarbonates, polyvinyl chloride (PVC), polyurethanes,polyethers, polyamides, polyimides, or copolymers of thesethermoplastics, such as PETG (glycol-modified polyethyleneterephthalate), among other polymeric plastic materials. In certainembodiments, the container is formed from a polyester, where polyestersof interest may include, but are not limited to, housings made ofpoly(alkylene terephthalates) such as poly(ethylene terephthalate)(PET), bottle-grade PET (a copolymer made based on monoethylene glycol,terephthalic acid, and other comonomers such as isophthalic acid,cyclohexene dimethanol, etc.), poly(butylene terephthalate) (PBT), andpoly(hexamethylene terephthalate); poly(alkylene adipates) such aspoly(ethylene adipate), poly(1,4-butylene adipate), andpoly(hexamethylene adipate); poly(alkylene suberates) such aspoly(ethylene suberate); poly(alkylene sebacates) such as poly(ethylenesebacate); poly(c-caprolactone) and poly([3-propiolactone);poly(alkylene isophthalates) such as poly(ethylene isophthalate);poly(alkylene 2,6-naphthalene-dicarboxylates) such as poly(ethylene2,6-naphthalene-dicarboxylate); poly(alkylene sulfonyl-4,4′-dibenzoates)such as poly(ethylene sulfonyl-4,4′-dibenzoate); poly(p-phenylenealkylene dicarboxylates) such as poly(p-phenylene ethylenedicarboxylates); poly(trans-1,4-cyclohexanediyl alkylene dicarboxylates)such as poly(trans-1,4-cyclohexanediyl ethylene dicarboxylate);poly(1,4-cyclohexane-dimethylene alkylene dicarboxylates) such aspoly(1,4-cyclohexane-dimethylene ethylene dicarboxylate);poly([2.2.2]-bicyclooctane-1,4-dimethylene alkylene dicarboxylates) suchas poly([2.2.2]-bicyclooctane-1,4-dimethylene ethylene dicarboxylate);lactic acid polymers and copolymers such as (S)-polylactide,(R,S)-polylactide, poly(tetramethylglycolide), andpoly(lactide-co-glycolide); and polycarbonates of bisphenol A,3,3′-dimethylbisphenol A, 3,3′,5,5′-tetrachlorobisphenol A,3,3′,5,5′-tetramethylbisphenol A; polyamides such as poly(p-phenyleneterephthalamide); Mylar™.

In some embodiments, the centrifuge activated valve is an umbrellavalve. In other embodiments, the centrifuge activated valve is a checkvalve. For example, the check valve may be a ball check valve, diaphragmcheck valve, lift check valve and a tilted disc check valve. In certainembodiments, the check valve is a ball and spring check valve, such as aball and spring valve having a stainless steel ball and spring.

In embodiments, the centrifuge activated valve is configured to open andclose in response to the force of centrifugation. In some embodiments,the valve opens in response to centrifugation. In other embodiments, thevalve closes immediately when centrifugation is stopped. In still otherembodiments, the valve gradually closes as centrifugation is slowed,where the valve takes a fully closed position when centrifugation isstopped.

In certain embodiments, the buoy has a first orifice at the base of theconcave outer surface at the buoy proximal end, a second orifice at aposition distal along the longitudinal axis of the buoy to the firstorifice and a channel that extends from the first orifice to the secondorifice. The first orifice and second orifice may be the same ordifferent shape, where examples of cross-sectional shapes include, butare not limited to rectilinear cross-sectional shapes, e.g., squares,rectangles, trapezoids, triangles, hexagons, etc., curvilinearcross-sectional shapes, e.g., circles, ovals, etc., as well as irregularshapes, e.g., a parabolic bottom portion coupled to a planar topportion, etc. The first orifice and the second orifice may also be thesame or different size and may range from 1 mm to 50 mm, such as from 2mm to 40 mm, such as from 3 mm to 30 mm and including from 5 mm to 25mm. The channel extending between the first orifice and the secondorifice may have the same or different cross-sectional dimensions as thefirst or second orifices. The channel may have the same cross-sectionalshape as the first orifice, the second orifice or may have a differentcross-sectional shape altogether. The length of the channel may alsovary, depending on the size of the buoy and amount of liquid samplebeing processed, e.g., ranging from 1 mm to 100 mm, such as from 2 mm to90 mm, such as from 3 mm to 80 mm, such as from 4 mm to 70 mm, such asfrom 5 mm to 60 mm and including from 10 mm to 50 mm.

In these embodiments, the buoy may also include a centrifuge activatedvalve (e.g., check valve) positioned at the first orifice, at the secondorifice or a position therebetween, where the centrifuge activated valveis configured with an open position and closed position where the checkvalve fluidically seals the second orifice in the closed position. Insome instances, the centrifuge activated valve is positioned at thefirst orifice such that in the closed position, the centrifuge activatedvalve fluidically seals the first orifice. In other instances, thecentrifuge activated valve is positioned at the second orifice such thatin the closed position, the centrifuge activated valve fluidically sealsthe second orifice. In still other instances, the centrifuge activatedvalve is positioned within the channel extending between the firstorifice and the second orifice and in the closed position, forms afluidic seal within the channel. For example, depending on the length ofthe channel, the centrifuge activated valve may be positioned 1 mm ormore from the first orifice, such as 2 mm or more, such as 5 mm or more,such as 10 mm or more, such as 25 mm or more, such as 50 mm or more andincluding 100 mm or more from the first orifice, such as beingpositioned from 1 mm to 100 mm from the first orifice, such as from 2 mmto 90 mm, such as from 3 mm to 80 mm, such as from 4 mm to 70 mm, suchas from 5 mm to 60 mm and including from 10 mm to 50 mm from the firstorifice. In certain embodiments, the centrifuge activated valve is inthe channel at a position that is equidistant from the first orifice andthe second orifice.

As discussed above, in certain embodiments, the centrifuge activatedvalve is configured to open in response to an applied force ofcentrifugation. Any convenient centrifuge activated valve (as describedabove) may be employed at the second orifice and may include, but arenot limited to check valves, such as ball check valves, umbrella valves,diaphragm check valves, lift check valves and a tilted disc checkvalves. In certain embodiments, the check valve is a mass and springvalve, such as a ball and spring check valve, including a ball andspring valve having a metal (e.g., stainless steel ball) and spring.

In embodiments, the centrifuge activated valve may be configured to openin response to the force of centrifugation. Depending on the type andsize of centrifuge activated valve, the valve may be configured to openin response to a force of centrifugation (in relative centrifugal force,RCF) ranging from 1 g to 50,000 g, such as from 2 g to 45,000 g, such asfrom 3 g to 40,000 g, such as from 5 g to 35,000 g, such as from 10 g to25,000 g, such as from 100 g to 20,000 g, such as from 500 g to 15,000 gand including from 1000 g to 10,000 g. As described above, subjectingthe sample to a force of centrifugation (e.g., centrifuging the subjectdevice with sample present in the container) is sufficient to open thevalve and collect one or more components of the sample on the buoy, suchas on the proximal end of the buoy, such as at the base of the concaveouter surface of the buoy, such as adjacent to an orifice on the buoy,such as in a channel in the buoy, such as on the surface of a centrifugeactivated suspension floor, such as on the surface of the ball in a balland spring valve. In one example where the multi-component liquid sampleis whole blood, subjecting the sample to a force of centrifugation issufficient to open the centrifuge activated valve and collect buffy coaton the surface of a centrifuge activated suspension floor, such as onthe surface of the ball in a ball and spring valve. In another example,the multi-component liquid sample is bone marrow aspirate and subjectingthe sample to a force of centrifugation is sufficient to open thecentrifuge activated valve and collect a component of the fractionatedbone marrow aspirate on the surface of a centrifuge activated suspensionfloor, such as on the surface of the ball in a ball and spring valve.

Depending on the cross-sectional shape of the buoy, the buoy has one ormore walls which extend along the longitudinal axis from the distal endto the proximal end of the buoy. The walls of the buoy, as described ingreater detail below, remain in close proximity to the interior walls ofthe container, during displacement of the buoy along the longitudinalaxis of the container. In certain embodiments, the walls of the buoy maybe configured to contact (i.e., touch) the inner walls of the containerduring displacement in response to the force of centrifugation. Thelength of the walls of the buoy may vary depending on the size of thebuoy as well as the size of the container. For example, the length ofthe walls of the buoy may range from 0.5 cm to 25 cm, such as from 1 cmto 22.5 cm, such as from 1.5 cm to 20 cm, such as from 2.5 cm to 17.5 cmand including from 5 cm to 15 cm.

In certain embodiments, the walls of the buoy may include one or moreribs. For example, the walls of the buoy may include 2 or more ribs,such as 3 or more ribs, such as 5 or more ribs, such as 10 or more ribsand including 25 or more ribs. Each rib may extend along a length of thebuoy by an amount that varies, such as extending along a length of thebuoy by 10% or more, such as by 25% or more, such as by 50% or more,such as by 75% or more, such as by 90% or more and including extendingentirely along the length of the walls of the buoy. Depending on thelength of the buoy, each rib may have a width which varies, ranging from0.1 mm to 10 mm, such as from 0.5 mm to 9.5 mm, such as from 1 mm to 9mm, such as from 2 mm to 8 mm and including a width from 3 mm to 5 mm,occupying between (but not including) 0 and 100% of the outer surfacearea of the buoy, and a length ranging from 1% to 100% of the length ofthe buoy.

In certain embodiments, the ribs are configured to reduce the sheer ofcomponents by the buoy during displacement along the longitudinal axisin response to the force of centrifugation. In other embodiments, theribs may be configured to maintain alignment within the container, suchas for example, where the walls of the buoy remain within 10° or less ofbeing parallel with the inner walls of the container during displacementof the buoy in response to the force of centrifugation, such as within7° or less, such as within 5° or less, such as within 3° or less, suchas within 2° or less, such as within 1° or less, such as within 0.5° orless, such as within 0.1° or less and including within 0.05° or less ofbeing parallel with the inner walls of the container during displacementof the buoy in response to the force of centrifugation. In certaininstances, ribs on the outer walls of the buoy are configured tomaintain alignment of the buoy such that the walls of the buoy remainparallel with the inner walls of the container during displacement ofthe buoy in response to the force of centrifugation. In certaininstances, ribs on the outer walls of the buoy are configured to collidewith protruding features or indentations on the inner wall of thecontainer in order to constrain or limit axial rotation of the buoywithin the container such that the buoy cannot make a full rotationwithout interference occurring. For example, the axial rotation of thebuoy may be limited to rotation by 25° or less, such as by 20° or less,such as by 15° or less, such as by 10° or less, such as by 5° or lessand including by 3° or less. In certain embodiments, ribs on the outerwalls of the buoy are configured to reduce axial rotation of the buoywithin the container by 50% or more as compared to a buoy without ribson the outer walls, such as by 75% or more, such as by 90% or more andincluding by 95% or more.

Depending on the size of the container, the cross-sectional dimensionsof the buoy may vary. For example, the cross-sectional dimensions of thebuoy may range from 0.5 cm to 25 cm, such as from 1 cm to 22.5 cm, suchas from 1.5 cm to 20 cm, such as from 2.5 cm to 17.5 cm and includingfrom 5 cm to 15 cm. Where the buoy has a cylindrical cross-section, thediameter may vary, in some embodiments, ranging from 1 cm to 10 cm, suchas from 2 cm to 9 cm, such as from 3 cm to 8 cm and including from 4 cmto 7 cm. Accordingly, the cross-sectional area of the buoy may vary,ranging from 1 to 500 cm², such as 5 to 250 cm², such as 10 to 200 cm²,such as 15 to 150 cm², such as 20 to 125 cm² and including from 25 to100 cm².

In embodiments, the buoy is configured to be displaced along thelongitudinal axis within the container. As such, the buoy is configuredto have a cross-section which is less than the cross-section of theinner cavity of the container. For example, the cross-section size ofthe buoy may be less than the cross-section of the inner cavity of thecontainer by 0.001 mm or more, such as by 0.005 mm or more, such as by0.01 mm or more, such as by 0.05 mm or more, such as by 0.1 mm or more,such as by 0.5 mm or more, such as by 1 mm or more and including by 2 mmor more. In other words, when the buoy is positioned inside thecontainer, there is space between the outer walls of the buoy and theinner walls of the container, such as a space ranging from 0.001 mm to 5mm, such as from 0.005 mm to 4.5 mm, such as from 0.01 mm to 4 mm, suchas from 0.05 mm to 3.5 mm, such as from 0.1 mm to 3 mm and, such as from0.5 mm to 2.5 mm and including from 1 mm to 2 mm of space between theouter walls of the buoy and the inner walls of the container.

Depending on the density of the multi-component liquid sample as well asthe components therein, the buoy may be displaced proximally ordisplaced distally during centrifugation. In some embodiments, the buoyis configured to have a density such that after centrifugation, the buoypositions at a particular location in the fractionated sample. Forexample, the buoy may be configured to have a density such that aftercentrifugation, the buoy is positioned at the interface between twofractionated components. In other embodiments, the buoy may beconfigured to have a density such that after centrifugation, the buoy ispositioned within a predetermined fraction, such as within a bottommostfraction, such as within an uppermost fraction or within some fractionin between. In yet other embodiments, the buoy may be configured to havea density such that after centrifugation, the buoy is positioned at thebottom of the container. In still other embodiments, the buoy may beconfigured to have a density such that after centrifugation, the buoy ispositioned at the top of the container. Depending on the multi-componentliquid sample, the buoy has a density which varies, ranging from 0.1g/mL to 2 g/mL, such as from 0.2 g/mL to 1.95 g/mL, such as from 0.3g/mL to 1.9 g/mL, such as from 0.4 g/mL to 1.85 g/mL, such as from 0.5g/mL to 1.8 g/mL, such as from 0.6 g/mL to 1.75 g/mL, such as from 0.7g/mL to 1.7 g/mL, such as from 0.8 g/mL to 1.6 g/mL and including a buoydensity of from 1 g/mL to 1.5 g/mL and including a buoy density from1.04 g/mL to 1.10 g/mL. For example, the buoy may have a density thatranges from 1.01 g/mL to 1.2 g/mL, such as from 1.04 g/mL to 1.07 g/mLand including from 1.045 g/mL to 1.060 g/mL. In certain embodiments, thedensity of the buoy is 1.055 g/mL.

For example, in some instances the multi-component liquid sample is ablood or bone marrow aspirate sample and the buoy is configured to havea density such that after centrifugation, the buoy is positioned at aninterface between red blood cells and plasma. In other instances, thebuoy is configured to have a density such that after centrifugation, thebuoy is positioned a level in the fractionated blood sample occupied bythe buffy coat. In certain instances, the buoy is configured to have adensity that is greater than whole blood (density 1.06 g/mL) but lessthan red blood cells (density 1.09 g/mL to 1.11 g/ml), such as a densityranging from 1.061 g/mL to 1.09 g/mL.

The buoy may be formed from any suitable material including, but notlimited to, glass, metal or plastic, such as a flexible or rigidplastic, polymeric or thermoplastic materials. For example, suitablepolymeric plastics may include polycarbonates, polyvinyl chloride (PVC),polyurethanes, polyethers, polyamides, polyimides, or copolymers ofthese thermoplastics, such as PETG (glycol-modified polyethyleneterephthalate), among other polymeric plastic materials. In certainembodiments, the container is formed from a polyester, where polyestersof interest may include, but are not limited to, housings made ofpoly(alkylene terephthalates) such as poly(ethylene terephthalate)(PET), bottle-grade PET (a copolymer made based on monoethylene glycol,terephthalic acid, and other comonomers such as isophthalic acid,cyclohexene dimethanol, etc.), poly(butylene terephthalate) (PBT), andpoly(hexamethylene terephthalate); poly(alkylene adipates) such aspoly(ethylene adipate), poly(1,4-butylene adipate), andpoly(hexamethylene adipate); poly(alkylene suberates) such aspoly(ethylene suberate); poly(alkylene sebacates) such as poly(ethylenesebacate); poly(E-caprolactone) and poly(p-propiolactone); poly(alkyleneisophthalates) such as poly(ethylene isophthalate); poly(alkylene2,6-naphthalene-dicarboxylates) such as poly(ethylene2,6-naphthalene-dicarboxylate); poly(alkylene sulfonyl-4,4′-dibenzoates)such as poly(ethylene sulfonyl-4,4′-dibenzoate); poly(p-phenylenealkylene dicarboxylates) such as poly(p-phenylene ethylenedicarboxylates); poly(trans-1,4-cyclohexanediyl alkylene dicarboxylates)such as poly(trans-1,4-cyclohexanediyl ethylene dicarboxylate);poly(1,4-cyclohexane-dimethylene alkylene dicarboxylates) such aspoly(1,4-cyclohexane-dimethylene ethylene dicarboxylate);poly([2.2.2]-bicyclooctane-1,4-dimethylene alkylene dicarboxylates) suchas poly([2.2.2]-bicyclooctane-1,4-dimethylene ethylene dicarboxylate);lactic acid polymers and copolymers such as (S)-polylactide,(R,S)-polylactide, poly(tetramethylglycolide), andpoly(lactide-co-glycolide); and polycarbonates of bisphenol A,3,3′-dimethylbisphenol A, 3,3′,5,5′-tetrachlorobisphenol A,3,3′,5,5′-tetramethylbisphenol A; polyamides such as poly(p-phenyleneterephthalamide); Mylar™

As summarized above, devices for separating components of amulti-component liquid sample according to certain embodiments include acontainer configured with a buoy that can be displaced along alongitudinal axis within the container. The container has a distal endand a proximal end with walls between the distal end and proximal endthat together form an inner cavity within the container such that thebuoy can freely be displaced along the longitudinal axis of thecontainer during centrifugation without resistance by the walls of thecontainer. In some embodiments, the outer walls of the container andinner cavity have the same cross-sectional shape where cross-sectionalshapes of interest include, but are not limited to curvilinearcross-sectional shapes, e.g., circles, ovals, rectilinear crosssectional shapes, e.g., squares, rectangles, trapezoids, triangles,hexagons, etc., as well as irregular shapes, e.g., a parabolic bottomportion coupled to a planar top portion. For example, both the outerwalls of the container and the inner cavity may have circular or ovalcross sections or both the outer walls of the container and the innercavity may have polygonal (e.g., octagonal) cross sections. In otherembodiments, the outer walls and inner cavity of the container havedifferent cross-sectional shapes (e.g., container having a polygonalcross-section and inner chamber having a circular cross-section). Incertain embodiments, the container is a tube and the cross-sectionalshape the outer walls and the inner walls are both circular.

The size of the inner cavity of the container may vary, where in someinstances the length of the inner cavity of the container may range from1 cm to 25 cm, such as from 2.5 cm to 22.5 cm, such as from 5 cm to 20cm, such as from 7.5 cm to 17.5 cm and including from 10 cm to 15 cm andthe width of the inner cavity of the container may range from 1 cm to 20cm, such as from 2 cm to 17.5 cm, such as from 3 cm to 15 cm, such asfrom 4 cm to 12.5 cm and including from 5 cm to 10 cm. Where the innercavity of the container has a cylindrical cross-section, the diametermay vary, in some embodiments, ranging from 1 cm to 10 cm, such as from2 cm to 9 cm, such as from 3 cm to 8 cm and including from 4 cm to 7 cm.Accordingly, the volume of the container may vary, ranging from 1 to 500cm³, such as 5 to 250 cm³, such as 10 to 200 cm³, such as 15 to 150 cm³,such as 20 to 125 cm³ and including from 25 to 100 cm³. In someembodiments, the container of the subject separation devices is a tubehaving a volume ranging from 1 mL to 500 mL, such as from 2 mL to 400mL, such as from 3 mL to 300 mL, such as from 4 mL to 200 mL, such asfrom 5 mL to 150 mL and including from 10 mL to 100 mL.

In certain embodiments, the container may include one or more referenceidentifiers (i.e., markings), such as for measuring the volume of one ormore components of the sample or for providing guidance in removing apredetermine amount of the sample from the container (as described ingreater detail below). In some embodiments, the markings make referenceto volume, such as references in units of milliliters of sample. Incertain embodiments, the container includes one or more referenceidentifiers which provide for removal of a predetermined amount ofsample from the container, such as a removal of 10% or more of thesample, such as 25% or more of the sample, such as 50% or more of thesample, such as 75% or more of the sample and including 90% or more ofthe sample. In other embodiments, the container includes one or morereference identifiers which provide for removal of a predeterminedamount of a particular separated fraction from the centrifuged sample,such as 10% or more from a particular fraction, such as 25% or more froma particular fraction, such as 50% or more from a particular fraction,such as 75% or more from a particular fraction and including 90% or morefrom a particular fraction. For example, in certain embodiments, themulti-component liquid sample is whole blood or bone marrow aspirate andcontainers of interest include one or more reference identifiers whichprovide for removal of a predetermined amount of the plasma fraction ofthe centrifuged whole blood sample, such as 10% or more of the plasmafraction, such as 25% or more of the plasma fraction, such as 50% ormore of the plasma fraction, such as 75% or more of the plasma fractionand including 90% or more of the plasma fraction. Any suitable type ofmarking on the container may be used, such as for example, printedmarkings on the inside or outside of the container, or markings whichare etched into the container walls.

The container may be formed from any suitable material including, butnot limited to, glass, metal or plastic, such as a flexible or rigidplastic, polymeric or thermoplastic materials. For example, suitablepolymeric plastics may include polycarbonates, polyvinyl chloride (PVC),polyurethanes, polyethers, polyamides, polyimides, or copolymers ofthese thermoplastics, such as PETG (glycol-modified polyethyleneterephthalate), among other polymeric plastic materials. In certainembodiments, the container is formed from a polyester, where polyestersof interest may include, but are not limited to, housings made ofpoly(alkylene terephthalates) such as poly(ethylene terephthalate)(PET), bottle-grade PET (a copolymer made based on monoethylene glycol,terephthalic acid, and other comonomers such as isophthalic acid,cyclohexene dimethanol, etc.), poly(butylene terephthalate) (PBT), andpoly(hexamethylene terephthalate); poly(alkylene adipates) such aspoly(ethylene adipate), poly(1,4-butylene adipate), andpoly(hexamethylene adipate); poly(alkylene suberates) such aspoly(ethylene suberate); poly(alkylene sebacates) such as poly(ethylenesebacate); poly(£-caprolactone) and poly([3-propiolactone);poly(alkylene isophthalates) such as poly(ethylene isophthalate);poly(alkylene 2,6-naphthalene-dicarboxylates) such as poly(ethylene2,6-naphthalene-dicarboxylate); poly(alkylene sulfonyl-4,4′-dibenzoates)such as poly(ethylene sulfonyl-4,4′-dibenzoate); poly(p-phenylenealkylene dicarboxylates) such as poly(p-phenylene ethylenedicarboxylates); poly(trans-1,4-cyclohexanediyl alkylene dicarboxylates)such as poly(trans-1,4-cyclohexanediyl ethylene dicarboxylate);poly(1,4-cyclohexane-dimethylene alkylene dicarboxylates) such aspoly(1,4-cyclohexane-dimethylene ethylene dicarboxylate);poly([2.2.2]-bicyclooctane-1,4-dimethylene alkylene dicarboxylates) suchas poly([2.2.2]-bicyclooctane-1,4-dimethylene ethylene dicarboxylate);lactic acid polymers and copolymers such as (S)-polylactide,(R,S)-polylactide, poly(tetramethylglycolide), andpoly(lactide-co-glycolide); and polycarbonates of bisphenol A,3,3′-dimethylbisphenol A, 3,3′,5,5′-tetrachlorobisphenol A,3,3′,5,5′-tetramethylbisphenol A; polyamides such as poly(p-phenyleneterephthalamide); Mylar™.

Depending on the type of container employed, the opacity of thecontainer to visible light may vary. In some embodiments, containers ofinterest are transparent. In other embodiments, containers aretranslucent to visible light. In yet other embodiments, containers areopaque to visible light.

In some embodiments, containers of the subject separation devices alsoinclude a cap configured to close off the proximal end of the container.For example, the cap may be a screw cap, a snap-on cap or a cap whichconnects the container by a permanent, semi-permanent or non-permanentadhesive. In certain instances, the cap forms a fluidic seal with thewalls of the container. The cap may be an integrated part of thecontainer, including where the cap is molded with, soldered, welded oraffixed to the container using a permanent adhesive. In otherembodiments, the cap is releasably attached to the container. By“releasably” is meant that the cap can be freely detached from andre-attached to the proximal end of the container. Where the cap isreleasably attached to the container, the cap may be non-permanentlyfastened to the container by any convenient attachment protocol,including but not limited to a hook and loop fastener, a latch, a notch,a groove, a pin, a tether, a hinge, Velcro, non-permanent adhesive, athreaded screw, or a combination thereof. In certain instances, thecontainer includes a threaded outer wall and is screw threaded with theinternal walls of the cap.

The cap may include one or more ports into the inner cavity of thecontainer, such as 2 or more ports, such as 3 or more ports, such as 4or more ports and including 5 or more ports. In certain embodiments, thecap includes only a single port. The ports may be any convenient portconfigured for fluidic or gaseous communication with the inner cavity ofthe container. In some embodiments, the cap includes a port forintroducing the multi-component liquid sample into the container or aport for collecting components of the liquid after centrifugation (asdescribed below). In certain embodiments, the cap includes a vent portconfigured to allow gas into and out of the container. In someinstances, the container includes a second opening in the cap to allowair to vent during sample introduction and removal. Where the capincludes a single port, the port is configured for both introducing themulti-component liquid sample into the container and for collecting oneor more components from the cavity of the container aftercentrifugation.

Any suitable port configuration may be employed depending on the desiredfunction of the port, where examples of ports include channels,orifices, channels having a check valve, a Luer taper fitting, a portwith a breakable seal (e.g., single use ports) among other types ofports. In some embodiments, the port is configured to connect to asyringe, such as for example to introduce a multi-component liquidsample (e.g., blood) into the container or to remove one or morecomponents from the container after centrifugation. In otherembodiments, the port is configured to facilitate access for a needleinto the cavity of the container to aspirate, mix and remove componentsfrom the container after centrifugation. In certain embodiments, theport is configured with a Luer taper fitting, such as a Luer-Lok or aLuer-slip.

Ports in the cap of the subject separation devices may be any suitableshape, where cross-sectional shapes of ports of interest include, butare not limited to rectilinear cross-sectional shapes, e.g., squares,rectangles, trapezoids, triangles, hexagons, etc., curvilinearcross-sectional shapes, e.g., circles, ovals, etc., as well as irregularshapes, e.g., a parabolic bottom portion coupled to a planar topportion. The dimensions of the ports may vary, in some embodimentsranging from 1 mm to 100 mm, such as from 2 mm to 95 mm, such as from 3mm to 90 mm, such as from 4 mm to 80 mm, such as from 5 mm to 70 mm,such as from 6 mm to 60 mm and including from 10 mm to 50 mm. In someembodiments, the port is a circular orifice and the diameter of the portranges from 1 mm to 100 mm, such as from 2 mm to 90 mm, such as from 4mm to 80 mm, such as from 5 mm to 70 mm, such as from 6 mm to 60 mm andincluding from 10 mm to 50 mm. Accordingly, depending on the shape ofthe ports, ports in the cap may have an opening which ranges from 0.01mm² to 250 mm², such as from 0.05 mm² to 200 mm², such as from 0.1 mm²to 150 mm², such as from 0.5 mm² to 100 mm², such as from 1 mm² to 75mm², such as from 2 mm² to 50 mm² and including from 5 mm² to 25 mm².

In some embodiments, one or more of the ports in the cap are configuredto be releasably attached to a syringe. For example, the cap may beconfigured to be non-permanently fastened to a syringe by a notch, agroove, a hook and loop fastener, Velcro, an adhesive, a threaded screwor a combination thereof. In some instances, the cap is configured to bereleasably attached to the syringe by inserting the syringe into theorifice of the port. In other instances, the cap is configured to bescrew threaded with the syringe. In yet other instances, the cap isconfigured with a Luer taper fitting (e.g., Luer-Lok, Luer slip, etc.)and the syringe is releasably attached to the port in the cap throughthe Luer taper fitting.

FIG. 1 illustrates an example of a device for separating components of amulti-component liquid (e.g., blood) according to certain embodiments.Device 100 includes a buoy 101 positioned inside of container 102. Buoy101 includes one or more sealed chambers 103 containing a gas andconcave outer surface 104 at the proximal end. At the base of theconcave outer surface of the buoy proximal end is a first orifice 105 influid communication with channel 106 and second orifice 107. Secondorifice 107 is sealed by ball-and-spring check valve 108 in the closedposition. Device 100 also includes a cap 109 positioned at the proximalend of the container. Cap 109 includes two ports, 109 a and 109 b. Port109 a is a vent port for gas flow into and out of the device. Port 109 bis an inlet/outlet for introducing multi-component liquid sample 110(e.g., blood) into container 100 or for removing one or more componentsafter separating the multi-component liquid sample by centrifugation, asdescribed below.

FIG. 2 illustrates an example of a device for separating components of amulti-component liquid according to another embodiment. Device 200includes buoy 201 positioned inside of container 202. At the base of theconcave outer surface of the buoy proximal end is a first orifice 205 influid communication with channel 206 and second orifice 207. Secondorifice 207 is adjacent to ball-and-spring check valve 108 in the openposition. Buoy 201 includes one or more sealed chambers 203 containing agas and concave outer surface 204 at the proximal end. Device 200 alsoincludes a cap 209 positioned at the proximal end of the container whichincludes two ports, vent port 209 a for gas flow into and out of thedevice and inlet/outlet port 209 b for introducing multi-componentliquid sample 210 (e.g., blood) into container 200 or for removing oneor more components after separating the multi-component liquid sample bycentrifugation.

In certain embodiments, the container also includes a conduit thatextends from one or more of the ports in the cap to the proximal end ofthe buoy. In these embodiments, the port in the cap is in fluidcommunication with the proximal end of the buoy through the conduit. Putanother way, the conduit includes two openings, a first opening in fluidcommunication with the port in the cap and a second opening in fluidcommunication with the proximal end of the buoy. The conduit may beintegrated with or may be releasably attached to one or more of the portand the proximal end of the buoy. In some embodiments, the conduit isintegrated with both the port in the cap and the buoy proximal end. Theconduit may be integrated such as by co-molding, soldering, welding oraffixing the conduit using a permanent adhesive. In other embodiments,the conduit is releasably attached to both the port in the cap and theproximal buoy end. The conduit may be releasably attached such as bynon-permanently fastening with a notch, groove, snap-on, hook and loopfastener, Velcro, a threaded screw or with a nonpermanent adhesive. Inyet other embodiments, the conduit is integrated with the port in thecap and releasably attached to the proximal end of the buoy. In stillother embodiments, the conduit is releasably attached to the port in thecap and integrated with the proximal end of the buoy. Depending on thesize of the sample, the configuration of the conduit may vary. In someembodiments, the conduit is a linear tube extending from the port in thecap to the buoy proximal end. In other embodiments, the conduit isnon-linear. For example, the conduit may be curvilinear, circular,winding, coiled, twisted or have a helical configuration.

In certain embodiments, the conduit is flexible. The term “flexible” isused in its conventional sense to mean that the conduit is capable ofbeing bent without breaking or otherwise able to be turned, bowed, ortwisted, without breaking. In these embodiments, the conduit may bepliable and is not rigid or stiff. In other embodiments, the conduit isrigid. The term “rigid” is used in its conventional sense to mean thatthe conduit is stiff and not capable of substantially being bent withoutbreaking.

Depending on the chemical constitution of specific conduits employed,the durometer hardness of conduits of interest may vary. In certainembodiments, the durometer hardness of conduits ranges from 10 Shore 00to 100 Shore 00, such as 20 Shore 00 to 90 Shore 00, such as 30 Shore 00to 80 Shore 00 and including 40 Shore 00 to 70 Shore 00. In otherembodiments, the durometer hardness of conduits of interest ranges from10 Shore A to 100 Shore A, such as 20 Shore A to 90 Shore A, such as 30Shore A to 80 Shore A and including 40 Shore A to 70 Shore A.

The length of the conduit may vary, ranging from 1 cm to 100 cm, such asfrom 2 cm to 95 cm, such as from 3 cm to 90 cm, such as from 4 cm to 85cm, such as from 5 cm to 80 cm, such as from 6 cm to 75 cm, such as from7 cm to 70 cm, such as from 8 cm to 65 cm, such as from 9 cm to 60 cm,such and including from 10 cm to 50 cm. In embodiments, thecross-sectional shape of the conduit may vary, where examples ofcross-sectional shapes include, but are not limited to rectilinearcross-sectional shapes, e.g., squares, rectangles, trapezoids,triangles, hexagons, etc., curvilinear cross-sectional shapes, e.g.,circles, ovals, etc., as well as irregular shapes, e.g., a parabolicbottom portion coupled to a planar top portion, etc. The cross-sectionaldimensions of the conduit may range from 0.01 mm to 25 mm, such as from0.05 mm to 22.5 mm, such as from 0.1 mm to 20 mm, such as from 0.5 mm to17.5 mm, such as from 1 mm to 15 mm, such as from 2 mm to 12.5 mm, suchas from 3 mm to 10 mm and including from 5 mm to 10 mm. For example,where the conduit is a tube, the diameter of the conduit may range from0.01 mm to 25 mm, such as from 0.05 mm to 22.5 mm, such as from 0.1 mmto 20 mm, such as from 0.5 mm to 15 mm, such as from 1 mm to 10 mm andincluding from 3 mm to 5 mm.

As discussed above, the conduit extends from a port in the cap to thebuoy proximal end. The opening of the conduit may be positioned at anylocation on the proximal end of the buoy. For example, in someembodiments, the conduit opening is positioned adjacent to the base ofthe concave outer surface (e.g., adjacent to the orifice) of the buoyproximal end, such as 1 mm or more from the base of the outer concavesurface, such as 2 mm or more, such as 3 mm or more, such as 4 mm ormore and including 5 mm or more from the base of the outer concavesurface. In other embodiments, the conduit opening is positioned alongthe outer edge of the buoy proximal end, such as at a position adjacentto the inner wall of the container. In still other embodiments, theconduit opening is positioned between the outer edge of the buoyproximal end and the base of the concave outer surface of the buoyproximal end, such as 1 mm or more from the outer edge of the buoyproximal end, such as 2 mm or more, such as 3 mm or more, such as 4 mmor more, such as 5 mm or more and including being positioned 10 mm ormore from the outer edge of the buoy proximal end.

In certain embodiments, the opening of the conduit in fluidcommunication with the proximal end of the buoy includes a streammodulator. The stream modulator may be any suitable component which iscoupled to the opening of the conduit at the proximal end of the buoyand may be configured to regulate the output of fluid from the conduit(e.g., when introducing the multi-component fluid into the container orwhen reintroducing one or more fractions to the proximal end of thebuoy). The stream modulator may be a separate component which isattached to the conduit, such as with an adhesive or fastener or may befully integrated with the conduit, such as by co-molding, soldering orwelding the stream modulator to the conduit.

In embodiments, the stream modulator is in fluid communication with theproximal end of the buoy. In certain instances, the stream modulator isphysically attached (e.g., by an adhesive or other fastener) to theproximal end of the buoy. In certain embodiments, the stream modulatoris integrated directed into the buoy, such as where the stream modulatoris molded with, soldered, welded or affixed to the buoy using apermanent adhesive.

The stream modulator may be configured to regulate the output flow rateof fluid from the conduit to the proximal end of the buoy. For example,the stream modulator may be configured to increase the output flow rateof fluid from the conduit to the proximal end of the buoy, such as byincreasing the flow rate by 0.01 mL/second or more, such as by 0.1mL/second or more, such as by 1 mL/second or more, such as by 5mL/second or more, such as by 10 mL/second or more, such as by 25mL/second or more, such as by 50 mL/second or more, such as by 100mL/second or more, such as by 250 mL/second or more and including by 500mL/second or more. For example, the stream modulator may be configuredto increase the output flow rate of fluid from the conduit to theproximal end of the buoy by 1% or more, such as by 5% or more, such asby 10% or more, such as by 15% or more, such as by 25% or more, such asby 50% or more, such as by 75% or more and including increasing theoutput flow rate of fluid from the conduit to the proximal end of thebuoy by 90% or more. In other instances, the stream modulator may beconfigured to decrease the output flow rate of fluid from the conduit tothe proximal end of the buoy, such as by decreasing the flow rate by0.01 mL/second or more, such as by 0.1 mL/second or more, such as by 1mL/second or more, such as by 5 mL/second or more, such as by 10mL/second or more, such as by 25 mL/second or more, such as by 50mL/second or more, such as by 100 mL/second or more, such as by 250mL/second or more and including by 500 mL/second or more. For example,the stream modulator may be configured to decrease the output flow rateof fluid from the conduit to the proximal end of the buoy by 5% or more,such as by 10% or more, such as by 15% or more, such as by 25% or more,such as by 50% or more, such as by 75% or more and including decreasingthe output flow rate of fluid from the conduit to the proximal end ofthe buoy by 90% or more.

In other embodiments, the stream modulator is configured to regulate thepressure of fluid outputted from the conduit to the proximal end of thebuoy. In some instances, the stream modulator increases the pressure offluid outputted from the conduit to the proximal end of the buoy, suchas by 5% or more, such as by 10% or more, such as by 15% or more, suchas by 25% or more, such as by 50% or more, such as by 75% or more andincluding increasing the pressure of fluid outputted from the conduit tothe proximal end of the buoy by 90% or more. In other instances, thestream modulator decreases the pressure of fluid outputted from theconduit to the proximal end of the buoy, such as by 5% or more, such asby 10% or more, such as by 15% or more, such as by 25% or more, such asby 50% or more, such as by 75% or more and including decreasing thepressure of fluid outputted from the conduit to the proximal end of thebuoy by 90% or more.

The stream modulator may have an orifice having any convenient shape,depending on the desired shape of the outputted flow stream to theproximal end of the conduit. For example, the cross-sectional shape ofthe stream modulator orifice may include, but is not limited torectilinear cross-sectional shapes, e.g., squares, rectangles,trapezoids, triangles, hexagons, etc., curvilinear cross-sectionalshapes, e.g., circles, ovals, etc., as well as irregular shapes, e.g., aparabolic bottom portion coupled to a planar top portion, etc. Thestream modulator orifice may have the same dimensions as cross-sectionaldimensions of the conduit or may have different dimensions. In someembodiments, the orifice of the stream modulator has dimensions that arethe same as the cross-sectional dimensions of the conduit. In otherembodiments, the orifice of the stream modulator has differentdimensions from the cross-sectional dimensions of the conduit. In oneexample, the stream modulator has an orifice that is larger than thecross-sectional dimensions of the conduit. In another example, thestream modulator has an orifice that is smaller than the cross-sectionaldimensions of the conduit. For example, the orifice of the streammodulator may be 5% smaller or more than the cross-section of theconduit, such as 10% or more, such as 25% or more, such as 50% or moreand including 75% or more. In certain embodiments, the orifice of thestream modulator ranges from 0.01 mm to 25 mm, such as from 0.05 mm to22.5 mm, such as from 0.1 mm to 20 mm, such as from 0.5 mm to 17.5 mm,such as from 1 mm to 15 mm, such as from 2 mm to 12.5 mm, such as from 3mm to 10 mm and including from 5 mm to 10 mm.

In certain embodiments, the stream modulator may include one or moreprotrusions, such as a protrusion which directs fluid from the conduitto the orifice of the buoy. The protrusions may be physically coupled toone or more of the stream modulator and the proximal end of the buoy. Incertain instances, the protrusion for directing fluid from the conduitto the orifice of the buoy is incorporated into the stream modulator. Inother instances, the protrusion is affixed to the stream modulator witha fastener, such as with an adhesive, a latch, snap-fitted or with screwthreads.

FIG. 3A illustrates a three-dimensional view of an example of a devicefor separating components of a multi-component liquid according tocertain embodiments. Device 300 includes buoy 301 positioned inside ofcontainer 302. At the upper edge of the proximal end of the buoy is port303 connecting conduit 304 to the proximal end of the buoy 301. At thebase of the concave outer surface of buoy 301 proximal end is an orifice305 adjacent to ball-and-spring check valve 306 in the open position.Device 300 also includes a cap 307 positioned at the proximal end of thecontainer which includes two ports, vent port 307 a for gas flow intoand out of the device and inlet/outlet port 307 b connected to conduit304 for introducing a multi-component liquid sample (e.g., blood) intocontainer 300 or for removing one or more components after separatingthe multi-component liquid sample by centrifugation. Device 300 alsoincludes an angular position indicator 310 for indicating the angleposition of the device when placed on a support when collecting one ormore of the separated components (as described in greater detail below).

FIG. 3B illustrates a side view of an example of a device for separatingcomponents of a multi-component liquid according to certain embodiments.As described in FIG. 3A, device 300 a includes buoy 301 a positionedinside of container 302 a. At the upper edge of the proximal end of thebuoy is port 303 a connecting conduit 304 a to the proximal end of thebuoy 301 a. At the base of the concave outer surface of the buoyproximal end is an orifice 305 a adjacent to ball-and-spring check valvewhich includes ball 306 a and spring 306 b. In this example, buoy 301 aincludes a concave outer surface at the distal end.

FIGS. 3C-3D illustrate a three-dimensional perspective of an example ofa device having a multi-component liquid separated into two or moreregions (i.e., fractions) according to certain embodiments. Thecomponents of device 300 are similar to those as described above inFIGS. 3A and 3B. As shown in FIGS. 3C and 3D, the multicomponent liquid(e.g., blood) is separated into a plurality of fractions 315 a and 315b.

FIGS. 4A and 4B depict two different three-dimensional views of a devicefor separating components of a multi-component liquid according tocertain embodiments. FIG. 4A illustrates a bottom-view three-dimensionalperspective of device 400 a which includes buoy 401 a positioned insideof container 402 a that is covered by lid 403 a. The lid includes asingle self-sealing port 404 a for inputting a multicomponent sample andaspirating one or more fractions after centrifugation. Port 404 a is influid communication with the proximal end of buoy 401 a through conduit405 a. Buoy 401 a also includes ribs 407 a along the outer walls to aidin fluid bypass during displacement of the buoy along the longitudinalaxis of container 402 a during centrifugation. Buoy 401 a also includesa centrifuge activated ball and spring valve 406 a.

FIG. 4B illustrates a top-view three-dimensional perspective of device400 b which includes buoy 401 b positioned inside of container 402 bthat is covered by lid 403 b. Buoy 401 b has one or more sealed chambers408 b that can include a fluidic composition or a vacuum. Lid 403 bincludes a single port 404 b for inputting a multicomponent sample andaspirating one or more fractions. Port 404 b is in fluid communicationwith buoy 401 b through conduit 405 b which is connected to the proximalend of the buoy. Lid 403 b also includes an air vent 407 b. Ball andspring valve 406 b is positioned at the distal end of buoy 401 b andfluidically seals a second orifice at the bottom of channel 409 b.

FIGS. 4C and 4D depict two different side views of a device forseparating components of a multi-component liquid according to certainembodiments. FIG. 4C illustrates a side view of device 400 c beforecentrifugation where buoy 401 c is positioned at the bottom of container402 c. As described in greater detail below, during centrifugation thebuoy is displaced along the longitudinal axis of container 402 c (upwardas depicted in FIG. 4D). Buoy 401 c includes one or more sealed chambers407 c having a fluidic composition or containing a vacuum. The proximalend of buoy 401 c is in fluid communication with a single port 404 c inlid 403 c through conduit 405 c. To aid in resuspension of components onthe buoy (as described below), buoy 401 c includes a deflector 406 cwithin the bore in channel 408 c that is fluidically sealed by ball andspring valve 409 c. FIG. 4D illustrates a side view of device 400 dafter centrifugation where buoy 401 d has been displaced a distancealong the longitudinal axis from the bottom of container 402 d. Buoy 401d includes one or more sealed chambers 407 d having a fluidiccomposition or containing a vacuum. The proximal end of buoy 401 d is influid communication with a single port 404 d in lid 403 d throughconduit 405 d. As shown above in FIG. 4C, buoy 401 d also includes adeflector 406 d within the bore in channel 408 d to aid in resuspensionof components. Buoy 401 c also includes a ball and spring valve 409 d.

Methods for Separating Components by Centrifugation

As summarized above, aspects of the disclosure also include methods forseparating components of a multi-component liquid sample. Methodsaccording to certain embodiments include introducing a multi-componentliquid sample (e.g., blood) into a container of one or more of thesubject separation devices described above, subjecting the sample to aforce of centrifugation to produce two or more fractions in the sample,each fraction having a component from the sample of a different densityand collecting one or more components of the sample. The term“separating” is used herein in its conventional sense to refer to thephysical separation of a plurality of components based on a particularphysical or chemical property such as density of the component. Asdescribed in greater detail below, the multi-component liquid sample issubjected to a force of centrifugation for a duration sufficient tofractionate the components of the sample into two or more fractions(e.g., layers), each fraction containing components of differentdensity. In embodiments, components of the sample are separated suchthat each component has a higher concentration in a particular fraction(e.g., bottom layer, upper layer, middle layer, etc.) as compared to thesample before being subjected to the force of centrifugation. In otherwords, components of the multi-component liquid sample are fractionatedin a manner sufficient to enrich the components into particular layerswithin the liquid sample.

For example, the concentration of a component in a particular fractionof the sample (e.g., bottom layer, upper layer, middle layer, etc.) maybe increased by 5% or more, such as by 10% or more, such as by 20% ormore, such as by 25% or more, such as by 30% or more, such as by 50% ormore, such as by 75% or more, such as by 90% or more including by 95% ormore as compared to the sample before being subjected to the force ofcentrifugation. In some instances, the concentration of a component in aparticular region of the sample may be increased by 2-fold or more, suchas by 3-fold or more, such as by 5-fold or more, such as by 7-fold ormore and including by 10-fold or more.

In embodiments of the present disclosure, components of the sample maybe separated into two or more fractions such that 5% or more of acertain component is separated in a particular fraction (e.g., bottomlayer, upper layer, middle layer, etc.) of the sample, such as 10% ormore, such as 20% or more, such as 25% or more, such as 30% or more,such as 40% or more, such as 50% or more, such as 60% or more, such as70% or more, such as 80% or more, such as 90% or more, such as 95% ormore and including separating 99% or more of a component into aparticular fraction of the sample. In certain embodiments, 100% of thecomponent is separated into a particular fraction of the sample.

In one example, the sample is whole blood or a derivative thereof (e.g.,whole blood having one or more anticoagulants) and is subjected to aforce of centrifugation for a duration sufficient to separate 90% ormore of plasma into a first fraction, 90% or more of the buffy coat intoa second fraction and 90% or more of red blood cells into a thirdfraction. For instance, the whole blood sample or derivative thereof issubjected to a force of centrifugation for a duration sufficient toseparate 95% of the plasma into a first fraction, 95% of the buffy coatinto a second fraction and 95% of the red blood cells into a thirdfraction.

In another example, the sample is bone marrow aspirate or a derivativethereof and is subjected to a force of centrifugation for a durationsufficient to separate 90% or more of a first component of the bonemarrow aspirate into a first fraction, 90% or more of a second componentof the bone marrow aspirate into a second fraction and 90% or more of athird component of the bone marrow aspirate into a third fraction. Forinstance, the bone marrow aspirate or derivative thereof is subjected toa force of centrifugation for a duration sufficient to separate 95% ormore of a first component of the bone marrow aspirate into a firstfraction, 95% or more of a second component of the bone marrow aspirateinto a second fraction and 95% or more of a third component of the bonemarrow aspirate into a third fraction.

As discussed above, the term multi-component liquid sample is used todescribe suspended media having more than one component and may include,but is not limited to biological samples. Biological samples may includea whole organism, plant, fungi or a subset of animal tissues, cells orcomponent parts which may in certain instances be found in blood, mucus,lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva,bronchoalveolar lavage, amniotic fluid, amniotic cord blood, urine,vaginal fluid and semen. As such, a “biological sample” refers to boththe native organism or a subset of its tissues as well as to ahomogenate, lysate or extract prepared from the organism or a subset ofits tissues, including but not limited to, for example, plasma, serum,spinal fluid, lymph fluid, sections of the skin, respiratory,gastrointestinal, cardiovascular, and genitourinary tracts, tears,saliva, milk, blood cells, tumors, organs. Biological samples mayinclude any type of organismic material, including both healthy anddiseased components (e.g., cancerous, malignant, necrotic, etc.). Incertain embodiments, the biological sample is a liquid sample, such aswhole blood or derivative thereof, bone marrow aspirate, stromalvascular fraction, plasma, tears, sweat, urine, semen, etc., where insome instances the sample is a blood sample, including whole blood, suchas blood obtained from venipuncture or fingerstick (where the blood mayor may not be combined with any reagents prior to assay, such aspreservatives, anticoagulants, etc.). The term “blood sample” refers towhole blood or a subset of blood components, including but not limitedto platelets, red blood cells, white cells and blood plasma. In someembodiments, the blood sample is obtained from an in vivo source and caninclude blood samples obtained from tissues (e.g., cell suspension froma tissue biopsy, cell suspension from a tissue sample, etc.) or directlyfrom a subject. In some cases, blood samples derived from a subject arecultured, stored, or manipulated prior to evaluation.

In certain embodiments the source of the biological sample is a “mammal”or “mammalian”, where these terms are used broadly to describe organismswhich are within the class mammalia, including the orders carnivore(e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), andprimates (e.g., humans, chimpanzees, and monkeys). In some instances,the subjects are humans. The methods may be applied to samples obtainedfrom human subjects of both genders and at any stage of development(i.e., neonates, infant, juvenile, adolescent, adult), where in certainembodiments the human subject is a juvenile, adolescent or adult. Whilethe present disclosure may be applied to samples from a human subject,it is to be understood that the methods may also be carried-out onsamples from other animal subjects (that is, in “non-human subjects”)such as, but not limited to, birds, mice, rats, dogs, cats, livestockand horses.

In embodiments, the multi-component liquid sample may also be abiological sample (as described above) that includes one or morecompounds, such as a preservative, antioxidant, stabilizer, surfactant,anticoagulant, chelating agent and the like. In certain instances, themulti-component liquid sample is whole blood or bone marrow aspiratethat includes one or more anticoagulants. For example, themulti-component liquid sample may be whole blood or bone marrow aspiratethat contains heparin or a calcium chelating agent (e.g., citrate orEDTA). The concentration of each compound in the biological sample mayvary depending on the type and volume of biological sample and may be0.001 mM or more, such as 0.005 mM or more, such as 0.01 mM or more,such as 0.05 mM or more, such as 0.1 mM or more, such as 0.5 mM or more,such as 1 mM or more, such as 5 mM or more, such as 10 mM or more, suchas 100 mM or more, such as 500 mM or more, such as 1000 mM or more andincluding 5000 mM or more. For example, the concentration of thecompounds in the biological sample may range from 0.001 mM to 5000 mM,such as from 0.01 mM to 1000 mM and including from 0.1 mM to 500 mM.

In practicing methods of the present disclosure, a multi-componentliquid sample is introduced into a container of one or more of thesubject devices (as described above) and subjected to a force ofcentrifugation for a duration sufficient to fractionate the componentsof the sample into two or more fractions (e.g., layers) within thesample. (e.g., higher density components forming a layer at the bottompart of the container and lower density components forming a layer atthe upper part of the container). One or more components are thencollected from the container.

In embodiments, the multi-component liquid sample may be introduced intothe container by any convenient liquid dispensing protocol, such asintroducing the sample through one or more ports in the cap of thesubject devices with a pipette, a syringe with or without a needle, amanual or mechanical dispenser or computer-automated liquid dispensingprotocol. Where the container includes a single port (as describedabove), the multi-component liquid sample is introduced through thesingle port in the cap of the container. The volume of multi-componentsample introduced into the container varies depending on the type ofsample, size of device and amount of desired component recovery andranges from 1 mL to 5000 mL, such as from 5 mL to 4500 mL, such as from10 mL to 4000 mL, such as from 20 mL to 3500 mL, such as from 30 mL to3000 mL, such as from 40 mL to 2500 mL, such as from 50 mL to 2000 mL,such as from 75 mL to 1500 mL and including from 100 mL to 1000 mL.

In practicing the subject methods, the sample is subjected to a force ofcentrifugation one or more times. The term “force of centrifugation” isused herein in its conventional sense to refer to the force applied tothe sample through revolving the device about an axis of rotation wherethe force on the components of the sample is in certain embodiments,given by the relative centrifugal force (RCF). The force ofcentrifugation may be applied by any convenient protocol, where in someembodiments, the force of centrifugation is applied by centrifuging thedevice with introduced sample with a centrifuge. In these embodiments,any convenient centrifuge may be employed, such as for example afixed-angle centrifuge, a swinging bucket centrifuge, ultracentrifuge,solid bowl centrifuges, conical centrifuges, among other types ofcentrifuges. As described in greater detail below, the applied force ofcentrifugation (in relative centrifugal force, RCF) may vary dependingon the sample type and size and may range from 1 g to 50,000 g, such asfrom 2 g to 45,000 g, such as from 3 g to 40,000 g, such as from 5 g to35,000 g, such as from 10 g to 25,000 g, such as from 100 g to 20,000 g,such as from 500 g to 15,000 g and including from 1000 g to 10,000 g.

In some embodiments, the sample is subjected to the centrifugation forceimmediately after the sample is introduced into the subject separationdevice container. In other embodiments, the sample is subjected to thecentrifugation force a predetermined period of time after introducingthe sample into the device container. For example, the sample may besubjected to the centrifugation force 0.01 minutes or more afterintroducing the sample into the device container, such as after 0.05minutes or more, such as after 0.1 minutes or more, such as after 0.5minutes or more, such as after 1 minute or more, such as after 5 minutesor more, such as after 10 minutes or more, such as after 15 minutes ormore, such as after 30 minutes or more and including 60 minutes afterintroducing the sample into the device container.

In certain embodiments, methods include a storage or prefabrication stepwhere the sample is a specimen that has been preloaded into one or moreof the subject separation devices and stored for a predetermined periodof time before subjecting the sample to the centrifugation force. Theamount of time the sample is preloaded and stored may vary, such as 0.1hours or more, such as 0.5 hours or more, such as 1 hour or more, suchas 2 hours or more, such as 4 hours or more, such as 8 hours or more,such as 16 hours or more, such as 24 hours or more, such as 48 hours ormore, such as 72 hours or more, such as 96 hours or more, such as 120hours or more, such as 144 hours or more, such as 168 hours or more andincluding preloading the sample for 240 hours or more. For example, theamount of time the sample is preloaded and stored may range from 0.1hours to 240 hours, such as from 0.5 hours to 216 hours, such as from 1hour to 192 hours and including preloading the sample from 5 hours to168 hours before subjecting the sample to the centrifugation force. Forinstance, the sample may be preloaded into one or more of the subjectseparation devices at a remote location (e.g., as in a physician'soffice or outpatient clinic) and sent to a laboratory for processing inaccordance with the subject methods. By “remote location” is meant alocation other than the location at which the sample is obtained andpreloaded. For example, a remote location could be another location(e.g., office, lab, etc.) in the same city, another location in adifferent city, another location in a different state, another locationin a different country, etc., relative to the location of the processingdevice, e.g., as described in greater detail below. In some instances,two locations are remote from one another if they are separated fromeach other by a distance of 10 m or more, such as 50 m or more,including 100 m or more, e.g., 500 m or more, 1000 m or more, 10,000 mor more, etc.

In embodiments, the sample is subjected to a force of centrifugation fora duration sufficient to separate components of different density intotwo or more fractions within the sample. The duration the sample issubjected to the force of centrifugation may vary and may be 0.01minutes or longer, such as for 0.05 minutes or longer, such as for 0.1minutes or longer, such as for 0.5 minutes or longer, such as for 1minute or longer, such as for 3 minutes or longer, such as for 5 minutesor longer, such as for 10 minutes or longer, such as for 15 minutes orlonger, such as for 20 minutes or longer, such as for 30 minutes orlonger, such as for 45 minutes or longer, such as for 60 minutes orlonger and including for 90 minutes or longer. For example, the samplemay be subjected to force of centrifugation for a duration which rangesfrom 0.01 minutes to 960 minutes, such as from 0.05 minutes to 480minutes, such as from 0.1 minutes to 240 minutes, such as from 0.5minutes to 120 minutes, such as from 1 minute to 90 minutes, such asfrom 5 minutes to 60 minutes and including from 10 minutes to 45minutes.

Depending on the volume of sample and density dispersity of the samplecomponents, the rotational speed of centrifugation may vary, such asfrom 1×10³ revolutions per minute (rpm) to 1000×10³ rpm, such as from2×10³ rpm to 900×10³ rpm, such as from 3×10³ rpm to 800×10³ rpm, such asfrom 4×10³ rpm to 700×10³ rpm, such as from 5×10³ rpm to 600×10³ rpm,such as from 10×10³ rpm to 500×10³ rpm and including from 25×10³ rpm to100×10³ rpm. The centrifuge may be maintained at a single speed or maybe changed to a different speed at any time during separation of thesample components. Where the centrifuge is operated at more than onespeed, the duration the centrifuge is maintained at each speed mayindependently be 0.01 minutes or more, such as 0.1 minutes or more, suchas 1 minute or more, such as 5 minutes or more, such as 10 minutes ormore, such as 30 minutes or more and including 60 minutes or more. Thetime period between each different speed employed may also vary, asdesired, being separated independently by a delay of 1 minute or more,such as 5 minutes or more, such as by 10 minutes or more, such as by 15minutes or more, such as by 30 minutes or more and including by 60minutes or more. In embodiments where the centrifuge is maintained atmore than two (i.e., three or more) speeds to subject the sample to thecentrifugation force, the delay between each speed employed may be thesame or different.

Depending on the type and number of components of different density inthe sample, the centrifuge may be maintained at a speed to subject thesample to the centrifugation force continuously or in discreteintervals. For example, in some embodiments, the centrifuge ismaintained at a speed to subject the sample to a centrifugation forcecontinuously. In other instances, the centrifuge is maintained at aspeed to subject the sample to a centrifugation force in discreteintervals, such as for example for intervals of for 0.01 minutes orlonger, such as for 0.05 minutes or longer, such as for 0.1 minutes orlonger, such as for 0.5 minutes or longer, such as for 1 minute orlonger, such as for 3 minutes or longer, such as for 5 minutes orlonger, such as for 10 minutes or longer, such as for 15 minutes orlonger, such as for 20 minutes or longer, such as for 30 minutes orlonger, such as for 45 minutes or longer, such as for 60 minutes orlonger and including for 90 minutes or longer. Where the centrifuge ismaintained at a speed in discrete intervals, methods may include 1 ormore intervals, such as 2 or more intervals, such as 3 or more intervalsand including 5 or more intervals.

The sample may be subjected to the force of centrifugation one or moretimes. In certain embodiments, methods include subjecting the sample toa force of centrifugation only one time. In other words, methodsaccording to this embodiment are characterized by a single applicationof the centrifugation force to the sample, such as by centrifuging thesubject device with introduced sample for a single spin interval. Inother embodiments, methods include subjecting the sample to a force ofcentrifugation 2 more times, such as 3 or more times, such as 4 or moretimes and including 5 or more times. Where the sample is subjected to aforce of centrifugation 2 or more times, the centrifugation force (e.g.,the speed of the centrifuge) and the duration may be the same ordifferent. In some embodiments, each time the sample is subjected to aforce of centrifugation, the centrifugation force and the duration isthe same. In other embodiments, the centrifugation force and theduration is different each time the sample is subjected to a force ofcentrifugation. In yet other embodiments, the centrifugation forceremains the same, but duration is different. In still other embodiments,the centrifugation force is different but duration remains the same.

In certain embodiments, methods include subjecting the sample to a forceof centrifugation in a two-step method where the sample is subjected toa first force of centrifugation when the centrifuge activated valve isin the open position and subjecting to a second force of centrifugationwhen the centrifuge activated valve is in the closed position. Therotational speed of centrifugation during each step may vary, asdescribed above, such as where the speed of centrifugation when thecentrifuge activated valve is in the open position ranges from 1×10³revolutions per minute (rpm) to 1000×10³ rpm (e.g., from 2×10³ rpm to500×10³ rpm) and the speed of centrifugation when the centrifugeactivated valve is in the closed position ranges from 1×10³ revolutionsper minute (rpm) to 1000×10³ rpm (e.g., from 2×10³ rpm to 500×10³ rpm).

The duration of centrifugation may also vary during each step, such assubjecting the sample to a force of centrifugation when the centrifugeactivated valve is in the open position for a duration ranging from 0.1minutes to 60 minutes (such as from 1 minute to 15 minutes) andsubjecting the sample to a force of centrifugation when the centrifugeactivated valve is in the closed position for a duration ranging from0.1 minutes to 60 minutes (such as from 0.5 minutes to 30 minutes).

In embodiments of the present disclosure, each step (introduction of thesample into the separation device container, subjecting the sample to acentrifugation force one or more times and collecting one or more of theseparated fractions of the sample) can be carried out at any suitabletemperature so long as the viability of the components (e.g., red bloodcells, white blood cells, platelets, etc.) of the sample are preservedas desired. As such, the temperature according to embodiments of thedisclosure may vary, such as from −80° C. to 100° C., such as from −75°C. to 75° C., such as from −50° C. to 50° C., such as from −25° C. to25° C., such as from −10° C. to 10° C., and including from 0° C. to 25°C.

Where necessary, the parameters for subjecting the samples to acentrifugation force may be changed at any time during methods of thepresent disclosure. For example, the speed of the centrifuge, theduration the sample is subjected to the centrifugation force and heatingor cooling of the sample may be changed one or more times during thesubject methods, such as two or more times, such as three or more timesand including five or more times.

In some embodiments, methods include changing the speed of thecentrifuge, such as by increasing or decreasing the speed by 1% or more,such as by 5% or more, such as by 10% or more, such as by 25% or more,such as by 50% or more, such as by 75% or more, such as by 90% or more,such as by 2-fold or more, such as by 5-fold or more, such as by 10-foldor more and including by 25-fold or more. For example, the speed of thecentrifuge may be increased or decreased by 0.5×10³ rpm or more, such asby 1×10³ rpm or more, such as by 2×10³ rpm or more, such as by 5×10³ rpmor more, such as by 10×10³ rpm or more, such as by 25×10³ rpm or moreand including increasing or decreasing the speed of the centrifuge by100×10³ rpm or more.

In other embodiments, the duration the sample is subjected to thecentrifugation force may be changed. For example, the duration thesample is subjected to the centrifugation force may be increased ordecreased by 0.01 minutes or longer, such as by 0.05 minutes or longer,such as by 0.1 minutes or longer, such as by 0.5 minutes or longer, suchas by 1 minute or longer, such as by 3 minutes or longer, such as by 5minutes or longer, such as by 10 minutes or longer, such as by 15minutes or longer, such as by 20 minutes or longer, such as by 30minutes or longer, such as by 45 minutes or longer, such as by 60minutes or longer and including by 90 minutes or longer.

In yet other embodiments, the temperature while subjecting the sample tothe centrifugation force may be changed. For example, the temperaturemay be raised or lower by 0.1° C. or more, such as by 0.5° C. or more,such as by 1° C. or more, such as by 2° C. or more, such as by 5° C. ormore and including raising or lowering the temperature by 8° C. or more.

In certain embodiments, methods include monitoring the centrifugedsample. Monitoring may include assessing (either by a human or with theassistance of a computer, if using a computer-automated processinitially set up under human direction) the extent of componentseparation within the sample. For example, monitoring separation ofcomponents by density into the two or more fractions within the samplemay include visually determining fraction boundaries between componentsof the sample. Monitoring separation of components may also includeassessing the physical and chemical properties of the components in eachfraction within the sample. Any convenient protocol can be employed tomonitor the sample, including but not limited to visual observation,laser scatter, fluorescence, phosphorescence, chemiluminescence, diffusereflectance, infrared spectroscopy, among other sensing protocols.

In some instances, monitoring includes collecting real-time data, suchas employing a detector (e.g., with a video camera). In other instances,monitoring includes assessing the sample at regular intervals, such asevery 0.01 minutes, every 0.05 minutes, every 0.1 minutes, every 0.5minutes, every 1 minute, every 5 minutes, every 10 minutes, every 30minutes, every 60 minutes or some other interval.

Methods of the present disclosure may also include a step of assessingthe sample to identify any desired adjustments to the subject protocol.In other words, methods in these embodiments include providing feedbackbased on monitoring the sample, where adjustments to the protocol mayvary in terms of goal, where in some instances the desired adjustment oradjustments that ultimately result in an improved fractionation ofcomponents by density within the sample, such as providing fasterseparation, improved purity or increased component enrichment of thecomponents into the two or more fractions within the sample.

Where feedback provided indicates that a particular protocol is lessthan optimal, such as where component separation requires too much timeor where component separation provides separated fractions withinsufficient enrichment (e.g., components of different density areundesirably mixed), methods may include changing one or more parts ofthe subject protocols. For example, one or more parameters forsubjecting the sample to a centrifugation force may be adjusted. In oneexample, methods include adjusting the speed of the centrifuge (asdescribed above). In another example, methods include changing(increasing or decreasing) the duration the sample is subjected to thecentrifugation force. In yet another example, methods include heating orcooling the sample.

As discussed above, in certain embodiments the buoy in the subjectdevices includes a centrifuge activated valve (e.g., ball and springvalve) that is configured to open and close in response to the force ofcentrifugation. For instance, the centrifuge activated valve may beconfigured to open in response to a force of centrifugation ranging from1 g to 50,000 g, such as from 2 g to 45,000 g, such as from 3 g to40,000 g, such as from 5 g to 35,000 g, such as from 10 g to 25,000 g,such as from 100 g to 20,000 g, such as from 500 g to 15,000 g andincluding from 1000 g to 10,000 g. In these embodiments, centrifugingthe device is sufficient to open the centrifuge activated valve andcollect one or more components of the sample on the surface of the buoy,such as on the proximal end of the buoy, such as at the base of theconcave outer surface of the buoy, such as adjacent to an orifice on thebuoy, such as in a channel in the buoy, such as on the surface of acentrifuge activated suspension floor of the centrifuge activated valve,such as on the surface of the ball in a ball and spring valve.Accordingly, methods according to certain embodiments of the presentdisclosure include subjecting the separation device to a force ofcentrifugation sufficient to open the centrifuge activated valve andcollect one or more components onto the surface of centrifuge activatedcheck valve. For instance, in one example where the multi-componentliquid sample is blood, subjecting the sample in the container of thesubject devices to a force of centrifugation is sufficient to open thecentrifuge activated valve and collect buffy coat on the surface of thebuoy, such as on the proximal end of the buoy, such as at the base ofthe concave outer surface of the buoy, such as adjacent to an orifice onthe buoy, such as in a channel in the buoy, such as on the surface ofthe centrifuge activated valve, such as on the surface of the ball in aball and spring valve.

In certain embodiments, the buoy in the subject devices displaces alongthe longitudinal axis within the container in response to the force ofcentrifugation. For example, the buoy may displace proximally ordistally during centrifugation. In some embodiments, the buoy isdisplaced along 25% or more of the length of the container, such as 35%or more, such as 50% or more, such as 60% or more, such as 75% or more,such as 90% or more, such as 95% or more, such as 97% or more andincluding 99% or more of the length of the container. In some instances,the buoy is positioned at the distal portion of the container and isdisplaced proximally along the longitudinal axis of the container inresponse to centrifugation. In certain instances, the buoy displacesduring centrifugation to a particular location in the fractionatedsample. In one example, the buoy displaces in response to centrifugationto a position at the interface between two fractionated components. Inanother example, the buoy displaces in response to centrifugation to aposition within a predetermined fraction, such as within a bottommostfraction, such as within an uppermost fraction or within some fractionin between. In yet another example, the buoy displaces in response tocentrifugation to a position at the bottom of the container. In stillanother example, the buoy displaces in response to centrifugation to aposition at the top of the container. In certain embodiments, the sampleis whole blood and methods include centrifuging the device withintroduced sample for a duration sufficient to displace the buoy fromthe bottom of the container to a position at the interface between redblood cells and plasma. In certain embodiments, the sample is bonemarrow aspirate and methods include centrifuging the device withintroduced sample for a duration sufficient to displace the buoy fromthe bottom of the container to a position at the interface between redblood cells and plasma so as to concentrate stem cells (e.g.,hematopoietic stem cells, mesenchymal stem cells, etc.). In certainembodiments, the sample is bone marrow aspirate and peripheral wholeblood combined and methods include centrifuging the device withintroduced sample for a duration sufficient to displace the buoy fromthe bottom of the container to a position at the interface between redblood cells and plasma so as to concentrate stem cells (e.g.,hematopoietic stem cells, mesenchymal stem cells, etc.). In certainembodiments, the sample is stromal vascular fraction derived fromadipose tissue and peripheral whole blood and centrifuging the devicewith introduced sample for a duration sufficient to displace the buoyfrom the bottom of the container to a position at the interface betweenred blood cells and plasma so as to concentrate stem cells (e.g.,mesenchymal cells) and endothelial cells from adipose together with theplatelets and monocytes derived from peripheral whole blood whiledepleting excess plasma and red blood cells.

In practicing the subject methods, one or more of the separatedfractions may be collected. In some embodiments, all of the separatedfractions are collected. Fractions may be collected using any suitablecollecting protocol, such as aspirating using a syringe with or withouta needle, a manual or mechanically operated serological pipette as wellas with an automated liquid collection system (e.g., acomputer-controlled collection apparatus). Where the sample is portionedinto two or more of the subject devices, collecting fractions mayinclude combining fractions of similar makeup. For example, where awhole blood or bone marrow aspirate sample is fractionated using two ormore of the subject devices, buffy coat from each of the devices may becollected (e.g., from the surface of a centrifuge activated suspensionfloor of the centrifuge activated valve on the buoy) and combined.

Separated fractions may be collected from the sample at any time aftersubjecting the sample to the force of centrifugation. In someembodiments, the separated fractions are collected 1 minute or greaterafter the separated fractions are prepared, such as 2 minutes orgreater, such as 3 minutes or greater, such as 5 minutes or greater,such as 10 minutes or greater and including 30 minutes or greater afterthe separated fractions are prepared.

In some embodiments, collecting one or more fractions from the sampleincludes removing a portion of a first fraction from the sample, mixingthe remaining portion of the first fraction with a second fractionwithin the buoy to produce a mixture of the first fraction and thesecond fraction and removing the mixture of the first fraction andsecond fraction from the container. In embodiments, removing a portionof the first fraction may include removal of 10% or more of the firstfraction, such as 15% or more, such as 25% or more, such as 50% or more,such as 75% or more and including removing 90% or more of the firstfraction. Put another way, methods according to these embodimentsinclude retaining 90% or less of the first fraction, such as 85% orless, such as 75% or less, such as 50% or less, such as 25% or less andincluding retaining 10% or less of the first fraction. For example,where the first fraction has a volume of 100 mL, methods may includeremoving 10 mL or more of the first fraction, such as 15 mL or more,such as 25 mL or more, such as 50 mL or more, such as 75 mL or more andincluding removing 90 mL or more of the first fraction. In certaininstances, the sample is whole blood and methods include removing 10% ormore of the platelet poor plasma, such as 15% or more, such as 25% ormore, such as 50% or more, such as 75% or more and including removing90% or more of the platelet poor plasma.

In certain embodiments, methods for removing a portion of the firstfraction include positioning a liquid collection device (e.g., needlewith syringe) a predetermined depth into the first fraction. Forexample, the liquid collection device may be positioned 1 mm or moreinto the first fraction, such as 2 mm or more, such as 3 mm or more,such as 5 mm or more, such as 10 mm or more and including 25 mm or moreinto the first fraction. In certain embodiments, the liquid collectiondevice is positioned to a depth as determined by one or more referenceindicators on the container. In still other embodiments, the liquidcollection device is positioned to a depth relative to the outer edge ofthe buoy proximal end, such as 1 mm or more above the outer edge of thebuoy proximal end, such as 2 mm or more, such as 3 mm or more, such as 5mm or more, such as 10 mm or more and including 25 mm or more above theouter edge of the buoy proximal end. In certain instances, methods forremoving a portion of the first fraction include positioning the liquidcollection device directly against the outer edge of the buoy proximalend.

In these embodiments, a second fraction is mixed with the remainingportion of the first fraction in the buoy. In one example, the remainingportion of the first fraction and the second fraction are mixed on theconcave outer surface of the proximal end of the buoy. In anotherexample, the remaining portion of the first fraction and the secondfraction are mixed on the surface of the centrifuge activated valve atthe base of the concave outer surface of the buoy proximal end. In yetanother example, where the buoy includes a channel above the centrifugeactivated valve (extending between a first orifice and second orifice,as described above), the remaining portion of the first fraction and thesecond fraction may be mixed within the channel in the buoy. In certaininstances, the sample is whole blood and methods include mixing theremaining portion of platelet poor plasma with buffy coat which collectson the surface of the buoy (such as in the channel when present or atthe base of the concave outer surface of the buoy proximal end when achannel is not present in the buoy) and producing platelet rich plasma.

The remaining portion of the first fraction and the second fraction maybe mixed in the buoy by any convenient protocol, such as for exampleagitation or stirring the two together. In certain embodiments, mixingthe remaining portion of the first fraction with the second fractionincludes aspirating the remaining portion of the first fraction intosyringe and reinjecting the first fraction into the buoy to mix thefirst fraction with the second fraction within the buoy. In otherembodiments mixing includes aspirating the remaining portion of thefirst fraction and the second fraction into a syringe and reinjectingboth the first fraction and second fraction into the buoy. In yet otherembodiments, methods include aspirating the remaining portion of thefirst fraction and the second fraction into a syringe, agitating thefirst fraction and second fraction in the syringe and reinjecting themixture into the buoy. Mixing the remaining portion of the firstfraction with the second fraction may be repeated as desired, such as 2or more times, such as 3 or more times, such as 4 or more times, such as5 or more times and including 10 or more times. After the remainingportion of first fraction and the second fraction are sufficiently mixedas desired (e.g., adequately mixed platelet rich plasma is produced),the mixture may be collected by any convenient liquid collectionprotocol, such as with a syringe with or without a needle, a manual ormechanically operated serological pipette as well as with an automatedliquid collection system (e.g., a computer-controlled collectionapparatus).

FIGS. 5A-5F illustrate step-by-step methods for separating components ofa multi-component liquid sample (e.g., whole blood) according to certainembodiments. FIG. 5A depicts device 500 having a buoy 501 positionedinside of container 502. Buoy 501 includes one or more sealed chambers503 containing a fluidic composition or a vacuum and concave outersurface 504 at the proximal end. At the base of the concave outersurface of the buoy proximal end is a first orifice 505 in fluidcommunication with channel 506 and second orifice 507. Second orifice507 is sealed by ball-and-spring check valve 508 in the closed position.Device 500 also includes a cap 509 positioned at the proximal end of thecontainer. Multi-component liquid sample 510 (e.g., blood) is introducedinto the container with a syringe or other suitable dispensing protocolthrough port 509 b in the cap with gas vent 509 a in an open or closedposition.

The beginning of centrifugation of the subject device with introducedsample is shown in FIG. 5B where the ball and spring valve closes thesecond orifice. After centrifugation for a sufficient duration and to adesired relative centrifugal force (as described above), the ball andspring valve proceeds to an open position as shown in FIG. 5C, such thatthere is fluid communication through second orifice 507. Aftersufficient duration, the sample is separated into fractions, such asinto a first fraction 511, a second fraction 512 and a third fraction513. For example, where the sample is blood, first fraction 511 may beplatelet poor plasma, second fraction 512 may be buffy coat, and thirdfraction 513 may be red blood cells. As shown here, second fraction 512(e.g., buffy coat) collects within channel 506 in response to theopening of valve 508 during centrifugation.

After centrifugation is complete, the desired components of the samplemay be collected. As shown in FIG. 5E, aspirating syringe 514 isinserted into the sample through port 509 b to a position at the outeredge of the buoy proximal end. A predetermined amount of fraction 511(e.g., platelet poor plasma) is removed. In this embodiment, theremaining portion of first fraction 511 is about the volume of theconcave outer surface of the buoy proximal end. The remaining portion offirst fraction 511 is aspirated into a second syringe 516 and reinjectedback into channel 506 to mix second fraction 512 (e.g., buffy coat) withthe remaining portion of first fraction 511 in the channel of the buoy.As shown in FIG. 5F, composition 515 (e.g., platelet rich plasma) whichis a mixture of first fraction 511 and second fraction 512 is thenremoved from the container.

As described above, in some embodiments the subject devices include acap positioned at the proximal end of the container and a conduit thatconnects one or more ports from the cap to the proximal end of the buoy.In some embodiments, collecting one or more components of the sample mayinclude the steps of: 1) positioning the container at a first angle(e.g., 20 degrees or more) with respect to an axis orthogonal to theground; 2) removing a portion of the first fraction of the samplethrough the conduit; 3) rotating the container by a second angle (e.g.,180 degrees) along the longitudinal axis of the container; 4) aspiratingthe remaining portion of the first fraction through the conduit; 5)mixing the remaining portion of the first fraction with a secondfraction within the buoy to produce a mixture of the first fraction andsecond fraction; and 6) removing the mixture of the first fraction andthe second fraction from the container through the conduit. In otherembodiments, collecting one or more components of the sample may includethe steps of: 1) positioning the container at a first angular positionwith respect to an axis orthogonal to the ground; 2) removing a portionof the first fraction of the sample through the conduit; 3) tilting thedevice to a second angular position with respect to the axis orthogonalto the ground; 4) aspirating the remaining portion of the first fractionthrough the conduit; 5) mixing the remaining portion of the firstfraction with a second fraction within the buoy to produce a mixture ofthe first fraction and second fraction; and 6) removing the mixture ofthe first fraction and the second fraction from the container throughthe conduit.

In these embodiments, removing a portion of the first fraction throughthe conduit includes removal of 10% or more of the first fraction, suchas 15% or more, such as 25% or more, such as 50% or more, such as 75% ormore and including removing 90% or more of the first fraction. As such,the container may be positioned at an angle that is sufficient to allowfor removal of the desired amount of the first fraction through theconduit, such as at an angle of 20 degrees or more relative to an axisorthogonal to the ground, such as 25 degrees or more, such as 30 degreesor more, such as 35 degrees or more, such as 45 degrees or more andincluding positioning the container at an angle that is 60 degrees ormore relative to an axis orthogonal to the ground. For example, wherethe sample is whole blood, methods may include removing 10% or more ofthe platelet poor plasma through the conduit by positioning thecontainer at an angle of 20 degrees or more relative to an axisorthogonal to the ground, such as 15% or more, such as 25% or more, suchas 50% or more, such as 75% or more and including removing 90% or moreof the platelet poor plasma.

The container may be positioned at the desired angle using any suitableprotocol, including but not limited to a manual support (e.g., stand) orwith a manual, mechanical or automated actuator. In some embodiments,the container may be coupled to a platform with a hinge or latch at aproximal edge of the container where the actuator positions the supportat an angle by raising the distal edge of the container. In otherembodiments, the actuator may be a lift column that is coupled to thebottom of the container and positioning of the container by the actuatorat an angle includes adjusting a pivot or rocker. In some instances,actuation of the container to the desired angle is carried out manually(i.e., positioning of the container by hand). In other instances, theactuator is a mechanical actuation device, such as for example amechanical lead screw assembly or a mechanically operated gearedtranslation device. In yet other embodiments, the actuator is amotor-driven displacement device, such as a motor actuated displacementstage, motor driven leadscrew assembly, motor-operated geared actuationdevice employing a stepper motor, servo motor, brushless electric motor,brushed DC motor, micro-step drive motor, high resolution stepper motor,among other types of motors.

In some embodiments, after removing a portion of the first fractionthrough the conduit, the container is rotated along the longitudinalaxis of the container (e.g., by 180 degrees) and the remaining portionof the first fraction is aspirated through the conduit and mixed withsecond fraction in the buoy. In one example, the remaining portion ofthe first fraction and the second fraction are mixed on the concaveouter surface of the proximal end of the buoy. In another example, theremaining portion of the first fraction and the second fraction aremixed on the surface of the centrifuge activated valve at the base ofthe concave outer surface of the buoy proximal end. In yet anotherexample, where the buoy includes a channel above the centrifugeactivated valve (extending between a first orifice and second orifice,as described above), the remaining portion of the first fraction and thesecond fraction may be mixed within the channel in the buoy. In certaininstances, the sample is whole blood and methods include mixing theremaining portion of platelet poor plasma with buffy coat which collectson the surface of the buoy (such as in the channel when present or atthe base of the concave outer surface of the buoy proximal end when achannel is not present in the buoy) and producing platelet rich plasma.

In other embodiments, after removing a portion of the first fraction,the container is tilted to a second angular position with respect to theaxis orthogonal to the ground and the remaining portion of the firstfraction of the biological sample is aspirated through the conduit andmixed with the second fraction in the buoy. The device may be tilted toany suitable angular position, depending on the position of the port inthe cap of the container and the volume of the biological sample and maybe 5° or more with respect to an axis orthogonal to the ground, such as10° or more, such as 15° or more, such as 25° or more, such as 30° ormore, such as 45° or more, such as 60° or more, such as 75° or more andincluding 80° or more. For example, the device may be tilted to a secondangular position that ranges from 1° to 90° with respect to an axisorthogonal to the ground, such as from 5° to 85°, such as from 10° to80°, such as from 15° to 75°, such as from 20° to 70° and including from3° to 60°. In other embodiments, the device is tilted to a secondangular position that is 5° or more with respect to the first angularposition, such as 10° or more, such as 15° or more, such as 25° or more,such as 30° or more, such as 45° or more, such as 60° or more, such as75° or more and including 80° or more. For example, the device may betilted to a second angular position that 1° to 90° with respect to thefirst angular position, such as from 5° to 85°, such as from 10° to 80°,such as from 15° to 75°, such as from 20° to 70° and including from 3°to 60°.

Mixing the remaining portion of the first fraction with the secondfraction may be repeated as desired, such as 2 or more times, such as 3or more times, such as 4 or more times, such as 5 or more times andincluding 10 or more times. After the remaining portion of firstfraction and the second fraction are sufficiently mixed as desired(e.g., adequately mixed platelet rich plasma is produced), the mixturemay be collected by any convenient liquid collection protocol, such aswith a syringe with or without a needle, a manual or mechanicallyoperated serological pipette as well as with an automated liquidcollection system (e.g., a computer-controlled collection apparatus).

In certain embodiments, the cap positioned at the proximal end of thecontainer includes a single port and the single port is connected to aconduit that fluidically couples the single port in the cap to theproximal end of the buoy. In these embodiments, collecting one or morecomponents of a separated multicomponent sample after centrifugationincludes the steps of: 1) positioning the container at a first anglewith respect to an axis orthogonal to the ground; 2) removing a portionof the first fraction of the separated multicomponent sample through theconduit; 3) rotating the container by a second angle along thelongitudinal axis of the container; 4) aspirating the remaining portionof the first fraction through the conduit; 5) mixing the remainingportion of the first fraction with a second fraction within the buoy toproduce a mixture of the first fraction and second fraction and 6)removing the mixture of the first fraction and the second fraction fromthe container through the conduit.

In these embodiments, removing a portion of the first fraction throughthe conduit includes removal of 10% or more of the first fraction, suchas 15% or more, such as 25% or more, such as 50% or more, such as 75% ormore and including removing 90% or more of the first fraction. Dependingon the position of the single port in the cap of the container, thecontainer is positioned at a first angle that is sufficient to allow forremoval of the desired amount of the first fraction through the singleport, such as at an angle that ranges from 10 degrees to 60 degreesrelative to an axis orthogonal to the ground, such as from 15 degrees to55 degrees, such as from 20 degrees to 50 degrees, such as from 25degrees to 45 degrees and including positioning the container at anangle which ranges from 30 degrees to 40 degrees relative to an axisorthogonal to the ground.

In some embodiments, positioning the container at the first angleincludes placing the container into an adjustable titter stand andadjusting the container to the desired angle (e.g., an angle rangingfrom 10 degrees to 60 degrees relative to an axis orthogonal to theground). For example, the container may be placed into the adjustabletilter stand and the container may be adjusted to the desired anglewhile visually monitoring the position of the conduit opening withrespect to the first fraction.

A portion of the first fraction of the separated multicomponent sampleis removed through the single port at the first angle. For example, 10%or more of the first fraction may be removed at the first angle, such as15% or more, such as 25% or more, such as 50% or more, such as 75% ormore and including removing 90% or more of the first fraction throughthe single port at the first angle. For example, where the sample iswhole blood, methods may include removing 10% or more of the plateletpoor plasma through the single port at the first angle, such as 15% ormore, such as 25% or more, such as 50% or more, such as 75% or more andincluding removing 90% or more of the platelet poor plasma.

In some embodiments, after removing a portion of the first fractionthrough the single port, the container is rotated by a second anglealong the longitudinal axis of the container and a second portion of thefirst fraction is aspirated through the single port. The second anglemay vary depending on the amount of the first fraction that remains inthe container and the position of the single port on the cap of thecontainer and may range from 90 degrees to 180 degrees, such as from 100degrees to 170 degrees, such as from 110 degrees to 160 degrees, such asfrom 120 degrees to 150 degrees and including from 130 degrees to 140degrees. In certain instances, the container is rotated 180 degreesalong the longitudinal axis of the container and the second portion ofthe first fraction is aspirated through the single port.

In other embodiments, after removing a portion of the first fractionthrough the single port, the container is tilted to a second angularposition with respect to the axis orthogonal to the ground and theremaining portion of the first fraction of the biological sample isaspirated through the single port. As discussed above, the container maybe tilted to any suitable angular position, depending on the position ofthe port in the cap of the container and the volume of the biologicalsample and may be 5° or more with respect to an axis orthogonal to theground, such as 10° or more, such as 15° or more, such as 25° or more,such as 30° or more, such as 45° or more, such as 60° or more, such as75° or more and including 80° or more. For example, the device may betilted to a second angular position that ranges from 1° to 90° withrespect to an axis orthogonal to the ground, such as from 5° to 85°,such as from 10° to 80°, such as from 15° to 75°, such as from 20° to70° and including from 3° to 60°. In other embodiments, the device istilted to a second angular position that is 5° or more with respect tothe first angular position, such as 10° or more, such as 15° or more,such as 25° or more, such as 30° or more, such as 45° or more, such as60° or more, such as 75° or more and including 80° or more. For example,the device may be tilted to a second angular position that ranges from1° to 90° with respect to the first angular position, such as from 5° to85°, such as from 10° to 80°, such as from 15° to 75°, such as from 20°to 70° and including from 3° to 60°.

Any suitable protocol may be used to position the container at the firstand second angles, including but not limited to a manual support (e.g.,stand) or with a manual, mechanical or automated actuator. In certainembodiments, the container is positioned at the first and second anglesusing a tilter stand which positions the container at one or more fixedangles (e.g., 20 degrees relative to an axis orthogonal to the ground)or may be an adjustable tilter stand where the user can adjust the firstand second angles as desired. In some instances, collection of eachportion of the first fraction as described above is carried out bymaintaining the container at the first and second angle by hand.

The second portion of the first fraction is mixed with a second fractionof the multicomponent sample at the buoy surface. For example, thesecond portion of the first fraction and the second fraction are mixedon the concave outer surface of the proximal end of the buoy. In anotherexample, the second portion of the first fraction and the secondfraction are mixed on the surface of the centrifuge activated valve atthe base of the concave outer surface of the buoy proximal end. In yetanother example, where the buoy includes a channel above the centrifugeactivated valve (extending between a first orifice and second orifice,as described above), the second portion of the first fraction and thesecond fraction may be mixed within the channel in the buoy. In certaininstances, the sample is whole blood and the second portion of the firstfraction is platelet poor plasma and the second fraction is buffy coatthat collects on the surface of the buoy (such as in the channel whenpresent or at the base of the concave outer surface of the buoy proximalend when a channel is not present in the buoy). In these embodiments,methods include aspirating the second portion of the platelet poorplasma at the second angle through the single port in the container capand reintroducing the second portion of the platelet poor plasma intothe container to mix with the buffy coat and produce platelet richplasma at the buoy.

FIGS. 6A-B illustrate an example of methods for separating components ofa multi-component liquid sample (e.g., whole blood) according to certainembodiments. FIG. 6A depicts device 600 having buoy 601 positionedinside of container 602. At the upper edge of the buoy proximal end ispositioned a port 603 a connecting to conduit 603. At the base of theconcave outer surface of the buoy proximal end is a first orifice 604with ball and spring check valve 605. Device 600 also includes a cap 609positioned at the proximal end of the container. Multi-component liquidsample (e.g., blood) is introduced into the container with a syringe orother suitable dispensing protocol through port 609 b in the cap withgas vent 609 a in an open or closed position. After removing device 600from the centrifuge, a first fraction 610 fills the upper portion ofcontainer 602. The container is positioned at an angle (e.g., 20 degreeswith respect to an axis orthogonal to the ground) and a portion of thefirst fraction 610 is removed by aspirating through the conduit which isin fluid communication with the first fraction through the port 603 a.After the amount of first fraction that can be removed through port 603a (i.e., the level of first fraction falls below the level of port 603a), a remaining portion of the first fluid remains in the container atthe surface of the buoy proximal end (FIG. 6B).

In certain embodiments, to remove the remaining portion of the firstfraction, the container is rotated 180 degrees along the longitudinalaxis of the container such that the remaining portion of the firstfraction is again in fluid communication with port 603 a. As describedabove, the remaining portion of the first fraction may be aspiratedthrough the conduit and reinjected to mix the remaining portion of thefirst fraction (e.g., platelet poor plasma) with a second fraction(e.g., buffy coat) which collects at the buoy proximal end.

FIGS. 7A-7H illustrate step-by-step methods for separating components ofa multi-component liquid sample (e.g., whole blood) according to certainembodiments. FIG. 7A depicts device 700 having a buoy 701 positionedinside of container 702. Buoy 701 includes one or more sealed chambers707. At the base of the proximal end of buoy 701 is a first orifice 706having a deflector plate 707 in fluid communication with channel 709 andsecond orifice 710. Second orifice 710 is sealed by ball and springvalve 715 in the closed position. Device 700 also includes a cap 703positioned at the proximal end of the container. The proximal end ofbuoy 701 is in fluid communication with a single port 704 in lid 703through conduit 705. Multi-component liquid sample 711 (e.g., blood) isintroduced into the container with a syringe or other suitabledispensing protocol through port 704 in lid 703.

After centrifugation for a sufficient duration and to a desired relativecentrifugal force (as described above), the sample is separated intofractions, such as into a first fraction 711 a, a second fraction 711 band a third fraction 711 c. For example, where the sample is wholeblood, first fraction 711 a may be platelet poor plasma, second fraction711 b may be buffy coat and third fraction 711 c may be packed red bloodcells. As shown here, second fraction 711 b (e.g., buffy coat) collectswithin channel 708 in response to the opening of valve 715 duringcentrifugation.

After centrifugation is complete, the desired components of the sampleare collected. To collect the desired components, device 700 is firstplaced into receptacle 701 b of tilter stand 700 b (described in greaterdetail below), aligned and clamped with locking lever 702 b (FIG. 7C).Next, receptacle 701 b is pivoted along groove 703 b of the tilter standto a first angular position (FIG. 7D). A portion of a first fraction ofthe sample (711 a) is aspirated through port 704 (via conduit 705) fromthe proximal end of buoy 701. After aspirating a portion of the firstfraction, the remaining portion of the first fraction remains in thedevice above the proximal end of the buoy (FIG. 7E). Next, receptacle701 b is pivoted along groove 703 b of the tilter stand to a secondangular position (FIG. 7F). The remaining portion of the first fractionthat remains above the proximal end of the buoy is aspirated throughport 704 (via conduit 705) at the second angular position andreintroduced back into the device to mix with second fraction 711 b atthe top of the centrifuge activated valve to produce a mixture of thefirst fraction and second fraction 711 ab (e.g., platelet rich plasma)(FIG. 7G). This mixture is then removed from the container through port704 (FIG. 7H).

FIG. 8 graphically illustrates an example of separating components of ablood sample according to certain embodiments. In step 1, an amount ofblood (55 mL with 5 mL of anticoagulant) is obtained or drawn into asyringe. The blood sample is transferred from a syringe into one or moreof the subject devices as described above through a port in the cap(step 2). As described above, the sample is subjected to a force ofcentrifugation by placing into a centrifuge (step 3). After removing thedevice from the centrifuge, a portion of the platelet poor plasma isremoved with an aspirating syringe (step 4). At step 5, the remainingportion of the platelet poor plasma is aspirated into a syringe andreinjected to rinse and mix the platelet poor plasma with the buffy coatwhich collects on the buoy (step 5). After sufficient mixing of theremaining portion of platelet poor plasma with buffy coat, the mixture(platelet rich plasma) is removed by aspirating into a syringe (step 6).

Systems for Separating by Centrifugation

Aspects of the present disclosure also include systems for practicingthe subject methods. As discussed above, methods for separatingcomponents according to embodiments of the present disclosure includeintroducing a multi-component liquid sample (e.g., blood) into acontainer of one or more of the subject separation devices describedabove, subjecting the sample to a force of centrifugation to produce twoor more fractions in the sample, each fraction having a component fromthe sample of a different density and collecting one or more componentsof the sample. In embodiments, systems are configured to apply a forceof centrifugation to the sample in the subject devices for a durationsufficient to fractionate the components of the sample into two or morefractions (e.g., layers), each fraction containing components ofdifferent density. Components of the sample are separated such that eachcomponent has a higher concentration in a particular fraction (e.g.,bottom layer, upper layer, middle layer, etc.) as compared to the samplebefore being subjected to the force of centrifugation. In other words,components of the multi-component liquid sample are fractionated in amanner sufficient to enrich the components into particular layers withinthe liquid sample.

In some embodiments, systems include one or more of the subject devicesdescribed above and a support for positioning the container at an anglewith respect to the axis orthogonal to the ground. For instance, thesupport may be configured to position and maintain the container at anangle that is 20 degrees or more with respect to the axis orthogonal tothe ground, such as 30 degrees or more, such as 45 degrees or more andincluding being configured to position and maintain the container at anangle that is 60 degrees with respect to the axis orthogonal to theground as 60 degrees or more. As such, the container may be positionedat an angle that is sufficient to allow for removal of the desiredamount of the first fraction through the conduit, such at an angle of 20degrees or more relative to an axis orthogonal to the ground, such as 25degrees or more, such as 30 degrees or more, such as 35 degrees or more,such as 45 degrees or more and including positioning the container at anangle that is 60 degrees or more relative to an axis orthogonal to theground.

The support may be any suitable support protocol, including but notlimited to a manual support (e.g., stand) or with a manual, mechanicalor automated actuator. In some embodiments, systems may include aplatform with a hinge or latch at a proximal edge of the container wherethe actuator positions the support at an angle by raising the distaledge of the container. In other embodiments, the support protocol is atilter stand configured to receive the device into a receptacle and toposition the device at any desired angular position. In otherembodiments, the actuator may be a lift column that is coupled to thebottom of the container and positioning of the container by the actuatorat an angle includes adjusting a pivot or rocker. In some instances,actuation of the container to the desired angle is carried out manually(i.e., positioning of the container by hand).

In other instances, the actuator is a mechanical actuation device, suchas for example a mechanical lead screw assembly or a mechanicallyoperated geared translation device. In yet other embodiments, theactuator is a motor-driven displacement device, such as a motor actuateddisplacement stage, motor driven leadscrew assembly, motor-operatedgeared actuation device employing a stepper motor, servo motor,brushless electric motor, brushed DC motor, micro-step drive motor, highresolution stepper motor, among other types of motors.

In some embodiments, the support includes a fastener for holding thedevice in the support in place. For example, fasteners may include, butare not limited to hook and loop fastener, a latch, a notch, a groove, apin, a tether, a hinge, Velcro, non-permanent adhesive, a threadedscrew, a dowel or a combination thereof.

FIGS. 9A-9B illustrate an example of a support for positioning one ormore of the subject devices described above at an angle with respect tothe axis orthogonal to the ground, where the angle may vary, ranging insome instances from 5 to 85°, such as 10 to 75°, including 15 to 50°,e.g., 15 to 40°, such as 15 to 30°, e.g., 20 to 25°. As shown in FIG.9A, the device is positioned in the support with the distal end in thesupport cradle with access to the inlet/outlet port. FIG. 9B illustratesthat the container of the subject device may be locked in place with afastener, such as a pin, dowel or other fasteners (as described above).

FIGS. 10A-10B illustrate an example of placing one or more of thesubject devices at an angle in a support to collect one or morecomponents of a separated multi-component liquid according to anotherembodiment. As shown in FIG. 10A, the device is positioned in the deviceat an angle with the distal portion of the container inserted into thesupport with access to inlet/outlet port. In this embodiment, theangular position indicator on the side of the device is lined up with amark on the support. FIG. 10B depicts an exploded view of the devicewhere the buoy port is at the lowest possible position (e.g., theconcave outer surface of the buoy proximal end slopes downward) whichfacilitates removal of the separated components of the multi-componentliquid.

FIGS. 11A-11C depict an adjustable tilter stand for positioning thedevice at a desired angle to remove one (or a portion) or more fractionsafter centrifugation according to certain embodiments as describedabove. FIGS. 11A and 11B show two different three-dimensionalperspectives of the tilter stand. Tilter stand 1100 includes areceptacle 1101 for holding and pivoting one of the subject devices.FIGS. 11A and 11B depict the receptacle at a first angle (90 degrees)with respect to an axis orthogonal to the ground. The device may besecured in receptacle 1101 with locking lever 1102. Tilter stand 1100includes groove 1103 configured to position receptacle 1101 at aplurality of angular positions. In certain embodiments, groove 1103includes one or more notches for positioning receptacle 1101 at discreetpositions (i.e., discreet angles with respect to an axis orthogonal tothe ground). In other embodiments, receptacle 1101 may be movedcontinuously along groove 1103 and secured at any desired angularposition, such as with a pin or screw. Tilter stand 1100 also includes aselector wheel 1105 which may be used to set one or more predeterminedpositions of the receptacle at a desired angle as receptacle 1101 ispivoted along groove 1103. Receptacle 1101 also includes a visualalignment slot 1104 to ensure proper placement of the device withinreceptacle 1101. The periphery of tilter stand 1100 may also include oneor more labels 1106 for predetermined positions for pivoting andsecuring receptacle 1101 along groove 1103 to collect one or morefractions from the subject devices (as described in greater detailbelow).

FIG. 11C depicts tilter stand 1100 from a front-facing perspective.Tilter stand 1100 includes receptacle 1101 for holding and pivoting oneof the subject devices. Devices placed in receptacle 1101 may be securedwith locking lever 1102. Devices positioned in receptacle 1101 arepivoted to the desired angular position along groove 1103. FIG. 11C alsoshows selector wheel 1105 to set one or more predetermined positions ofthe receptacle at a desired angle. Receptacle 1101 in FIG. 11C alsodepicts visual alignment slot 1104. One or more labels 1106 for pivotingand securing receptacle 1101 at predetermined angular positions tocollect one or more fractions from the subject devices may be included,in certain embodiments, along the periphery of tilter stand 1100.

In addition to one or more of the subject separation devices describedabove, systems of interest may also include a centrifuge for applying aforce of centrifugation to the sample. The term “centrifuge” is usedherein in its conventional sense to refer to an apparatus for rotatingone or more of the subject separation devices about a rotation axis toapply a centrifugal force to the components of the sample in the devicecontainer. Any convenient centrifuge protocol may be employed, includingbut not limited to fixed-angle centrifuges, swinging bucket centrifuges,ultracentrifuges, solid bowl centrifuges, conical centrifuges, amongother types of centrifuges. In certain embodiments, the centrifuge is acentrifuge with a horizontal rotor. In other embodiments, the centrifugeis a centrifuge with a fixed angle rotor. For example, the centrifugemay be certain instances a Horizon Model 755VES centrifuge (Drucker Co.,Port Matilda Pa.) having a horizontal rotor or fixed angle rotor andbrushless DC motor.

As described above, the subject centrifuges may be configured to apply aforce of centrifugation which varies, depending on the type of sample,size of separation device and desired separation of sample components.In embodiments, centrifuges of interest may apply a force ofcentrifugation which ranges (in relative centrifugal force, RCF) from 1g to 50,000 g, such as from 2 g to 45,000 g, such as from 3 g to 40,000g, such as from 5 g to 35,000 g, such as from 10 g to 25,000 g, such asfrom 100 g to 20,000 g, such as from 500 g to 15,000 g and includingfrom 1000 g to 10,000 g. Accordingly, centrifuges of interest may beconfigured to operate at rotation speeds which vary widely, such as from1×10³ revolutions per minute (rpm) to 1000×10³ rpm, such as from 2×10³rpm to 900×10³ rpm, such as from 3×10³ rpm to 800×10³ rpm, such as from4×10³ rpm to 700×10³ rpm, such as from 5×10³ rpm to 600×10³ rpm, such asfrom 10×10³ rpm to 500×10³ rpm and including from 25×10³ rpm to 100×10³rpm.

The centrifuge may also be a temperature-controlled centrifuge, wherethe temperature of the sample in the subject devices may be maintainedor changed (e.g., increased or decreased) as desired. For example, thecentrifuge may be configured to maintain the temperature of the samplein the subject devices from −80° C. to 100° C., such as from −75° C. to75° C., such as from −50° C. to 50° C., such as from −25° C. to 25° C.,such as from −10° C. to 10° C., and including from 0° C. to 25° C.

Centrifuges of interest may also be configured with monitoring protocolsfor assessing the sample during centrifugation. For example, thecentrifuge may include a viewing window to visually observecentrifugation or may include one or more sensors, such as laser scattersensors, fluorescence sensors, phosphorescence sensors,chemiluminescence sensors, diffuse reflectance sensors, infraredsensors, among other sensing protocols.

In certain embodiments, systems of interest further includecomputer-controlled systems for practicing the subject methods, wherethe systems may include one or more computers for automation orsemi-automation of a system for practicing methods described herein. Inthese embodiments, systems may include a computer having a computerreadable storage medium with a computer program stored thereon, wherethe computer program when loaded on the computer includes algorithm forcontrolling a liquid dispensing device to introduce a multi-componentliquid sample (e.g., blood) into a container of one or more of thesubject separation devices, algorithm for subjecting the sample to aforce of centrifugation to produce two or more fractions in the sampleand algorithm for controlling a liquid collection device to collect oneor more separated components of the sample. In certain embodiments, thecomputer program may also include algorithm for providing a blood samplefrom a sample source to the liquid dispensing device. For example, wherethe sample is a whole blood sample, the computer processor may alsoinclude algorithm for transferring the whole blood sample from a bloodcollection tube into the container of one or more of the subjectseparation devices.

In embodiments, the computer-controlled system includes an input moduleand a processing module. In some embodiments, the subject systems mayinclude an input module such that parameters or information about: 1)each sample including the type of sample (e.g., whole blood, a bloodderivative, citrated blood etc.), viscosity of the liquid sample, samplevolume and number of separated fractions expected from the sample; 2)components from the sample that are of interest; 3) desired speed of thecentrifuge for subjecting the sample to a force of centrifugation; 4)the temperature of the centrifuge and 5) the number of centrifugationintervals, etc. may be inputted into the computer. The processing moduleincludes memory having a plurality of instructions for performingcertain steps of the subject methods, such as introducing themulti-component sample into the container of the subject separationdevices, applying a force of centrifugation as well as instructions forcollecting the separated fractions from the sample.

The subject systems may include both hardware and software components,where the hardware components may take the form of one or moreplatforms, e.g., in the form of servers, such that the functionalelements, i.e., those elements of the system that carry out specifictasks (such as managing input and output of information, processinginformation, etc.) of the system may be carried out by the execution ofsoftware applications on and across the one or more computer platformsrepresented of the system.

Computer systems of interest may include a display and operator inputdevice. Operator input devices may, for example, be a keyboard, mouse,or the like. The processing module may include an operating system, agraphical user interface (GUI) controller, a system memory, memorystorage devices, and input-output controllers, cache memory, a databackup unit, and many other devices. The processor may be a commerciallyavailable processor or it may be one of other processors that are orwill become available. The processor executes the operating system andthe operating system interfaces with firmware and hardware in awell-known manner, and facilitates the processor in coordinating andexecuting the functions of various computer programs that may be writtenin a variety of programming languages, such as Java, Perl, C++, otherhigh level or low level languages, as well as combinations thereof, asis known in the art. The operating system, typically in cooperation withthe processor, coordinates and executes functions of the othercomponents of the computer. The operating system also providesscheduling, input-output control, file and data management, memorymanagement, and communication control and related services, all inaccordance with known techniques.

The system memory may be any of a variety of known or future memorystorage devices. Examples include any commonly available random accessmemory (RAM), magnetic medium such as a resident hard disk or tape, anoptical medium such as a read and write compact disc, flash memorydevices, or other memory storage device. The memory storage device maybe any of a variety of known or future devices, including a compact diskdrive, a tape drive, a removable hard disk drive, or a diskette drive.Such types of memory storage devices typically read from, and/or writeto, a program storage medium (not shown) such as, respectively, acompact disk, magnetic tape, removable hard disk, or floppy diskette.Any of these program storage media, or others now in use or that maylater be developed, may be considered a computer program product. Aswill be appreciated, these program storage media typically store acomputer software program and/or data. Computer software programs, alsocalled computer control logic, typically are stored in system memoryand/or the program storage device used in conjunction with the memorystorage device.

In some embodiments, a computer program product is described comprisinga computer usable medium having control logic (computer softwareprogram, including program code) stored therein. The control logic, whenexecuted by the processor the computer, causes the processor to performfunctions described herein. In other embodiments, some functions areimplemented primarily in hardware using, for example, a hardware statemachine. Implementation of the hardware state machine so as to performthe functions described herein will be apparent to those skilled in therelevant arts.

Memory may be any suitable device in which the processor can store andretrieve data, such as magnetic, optical, or solid state storage devices(including magnetic or optical disks or tape or RAM, or any othersuitable device, either fixed or portable). The processor may include ageneral purpose digital microprocessor suitably programmed from acomputer readable medium carrying necessary program code. Programmingcan be provided remotely to processor through a communication channel,or previously saved in a computer program product such as memory or someother portable or fixed computer readable storage medium using any ofthose devices in connection with memory. For example, a magnetic oroptical disk may carry the programming, and can be read by a diskwriter/reader. Systems of the invention also include programming, e.g.,in the form of computer program products, algorithms for use inpracticing the methods as described above. Programming according to thepresent invention can be recorded on computer readable media, e.g., anymedium that can be read and accessed directly by a computer. Such mediainclude, but are not limited to: magnetic storage media, such as floppydiscs, hard disc storage medium, and magnetic tape; optical storagemedia such as CD-ROM; electrical storage media such as RAM and ROM;portable flash drive; and hybrids of these categories such asmagnetic/optical storage media.

The processor may also have access to a communication channel tocommunicate with a user at a remote location. By remote location ismeant the user is not directly in contact with the system and relaysinput information to an input manager from an external device, such as acomputer connected to a Wide Area Network (“WAN”), telephone network,satellite network, or any other suitable communication channel,including a mobile telephone (e.g., smartphone) or tablet device.

Output controllers may include controllers for any of a variety of knowndisplay devices for presenting information to a user, whether a human ora machine, whether local or remote. If one of the display devicesprovides visual information, this information typically may be logicallyand/or physically organized as an array of picture elements. A graphicaluser interface (GUI) controller may include any of a variety of known orfuture software programs for providing graphical input and outputinterfaces between the system and a user, and for processing userinputs. The functional elements of the computer may communicate witheach other via system bus. Some of these communications may beaccomplished in alternative embodiments using network or other types ofremote communications. The output manager may also provide informationgenerated by the processing module to a user at a remote location, e.g.,over the Internet, phone or satellite network, in accordance with knowntechniques. The presentation of data by the output manager may beimplemented in accordance with a variety of known techniques. As someexamples, data may include SQL, HTML or XML documents, email or otherfiles, or data in other forms. The data may include Internet URLaddresses so that a user may retrieve additional SQL, HTML, XML, orother documents or data from remote sources. The one or more platformspresent in the subject systems may be any type of known computerplatform or a type to be developed in the future, although theytypically will be of a class of computer commonly referred to asservers. However, they may also be a main-frame computer, a workstation, or other computer type. They may be connected via any known orfuture type of cabling or other communication system including wirelesssystems, either networked or otherwise. They may be co-located or theymay be physically separated. Various operating systems may be employedon any of the computer platforms, possibly depending on the type and/ormake of computer platform chosen. Appropriate operating systems includeWindows NT®, Windows XP, Windows 7, Windows 8, iOS, Sun Solaris, Linux,OS/400, Compaq Tru64 Unix, SGI IRIX, Siemens Reliant Unix, and others.

Kits

Aspects of the invention further include kits, where kits include one ormore of the subject separation devices having a container and a buoyconfigured to be displaced along a longitudinal axis within thecontainer as described herein. In some instances, the kits can includeone or more additional components (e.g., buffers, water, solvent etc.).In some instances, the kits may further include a sample collectiondevice, e.g., blood collection device such as an evacuated bloodcollection tube, needle, syringe, pipette, tourniquet, etc. as desired.

The various assay components of the kits may be present in separatecontainers, or some or all of them may be pre-combined. For example, insome instances, one or more components of the kit, e.g., the separationdevice, containers, buoys, are present in a sealed pouch, e.g., asterile foil pouch or envelope.

In addition to the above components, the subject kits may furtherinclude (in certain embodiments) instructions for assembling the subjectkit components as well as for practicing the methods for separatingcomponents of a multi-component liquid sample as described herein. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, and the like. Yet another form of these instructions is acomputer readable medium, e.g., diskette, compact disk (CD), portableflash drive, and the like, on which the information has been recorded.Yet another form of these instructions that may be present is a websiteaddress which may be used via the internet to access the information ata removed site.

Utility

The subject devices, methods and systems find use in a variety ofapplications where it is desirable to separate components of amulti-component liquid sample. Embodiments of the present disclosurealso find use in purifying components of a biological sample, such aswhole blood and bone marrow aspirate where it is desirable to obtainisolated components of blood (e.g., white blood cells, stem cells, redblood cells, platelets, plasma, etc.) In some embodiments, the presentdisclosure finds use in preparing blood products having therapeuticapplications, such as platelet rich plasma. Embodiments also find use inthe preparation of samples from multi-component liquid where onlycertain components are desired, such as for laboratory assays,diagnostic tests or for other research applications.

In addition, applications of the present disclosure also find use wherecomponents (e.g., cells, proteins, polysaccharides or other largemacromolecular compound) prepared from a biological sample may bedesired for laboratory testing or for use in therapy. For example, thesubject devices and methods facilitate obtaining blood products that maybe used to treat wounds, accelerate tissue growth or other ailments,such as fistulas.

Notwithstanding the appended clauses, the disclosure set forth herein isalso defined by the following clauses:

1. A device for separating components of a multi-component liquid, thedevice comprising:

a container comprising a distal end and a proximal end; and

a buoy configured to be displaced along a longitudinal axis within thecontainer, wherein the buoy comprises one or more sealed chambers.

2. The device according to clause 1, wherein the buoy comprises afrustoconical shaped component.

3. The device according to clause 2, wherein the buoy comprises acylindrical proximal portion and frustoconical distal portion.

4. The device according to clause 2, wherein the proximal end of thebuoy comprises a concave outer surface.

7. The device according to clause 1, wherein the buoy has a density from1.01 g/mL to 1.2 g/mL.

8. The device according to clause 7, wherein the buoy has a density from1.04 g/mL to 1.07 g/mL.

9. The device according to clause 7, wherein the buoy has a density from1.045 g/mL to 1.060 g/mL.

10. The device according to clause 2, wherein the distal end of the buoycomprises a convex outer surface.

11. The device according to clause 2, wherein the distal end of the buoycomprises a concave outer surface.

11. The device according to clause 4, wherein the buoy comprises:

an orifice at the base of the concave outer surface; and

a centrifuge activated valve comprising an open position and a closedposition, wherein the valve is configured in the closed position tofluidically seal the orifice at the base of the concave outer surface.

12. The device according to clause 11, wherein the centrifuge activatedvalve is an umbrella valve.

13. The device according to clause 11, wherein the centrifuge activatedvalve is a check valve.

14. The device according to clause 13, wherein the centrifuge activatedvalve is selected from the group consisting of a ball check valve, adiaphragm check valve, a lift check valve and a tilted disc check valve.

15. The device according to clause 11, wherein the centrifuge activatedvalve comprises a ball and spring.

16. The device according to clause 15, wherein the ball comprises metal.

17. The device according to clause 15, wherein the ball comprisesstainless steel metal.

18. The device according to clause 4, wherein the buoy comprises:

a first orifice at the base of the concave outer surface;

a second orifice at a position distal along the longitudinal axis of thebuoy to the first orifice; and

a channel that extends from the first orifice to the second orifice;

a centrifuge activated valve comprising an open position and a closedposition, wherein the valve is configured in the closed position tofluidically seal the channel.

19. The device according to clause 18, wherein the centrifuge activatedvalve is positioned at the first orifice.

20. The device according to clause 18, wherein the centrifuge activatedvalve is positioned at the second orifice.

21. The device according to clause 18, wherein the centrifuge activatedvalve is positioned within the channel between the first orifice and thesecond orifice.

22. The device according to clause 18, wherein the centrifuge activatedvalve is an umbrella valve.

23. The device according to clause 18, wherein the centrifuge activatedvalve is a check valve.

24. The device according to clause 23, wherein the check valve isselected from the group consisting of a ball check valve, a diaphragmcheck valve, a lift check valve and a tilted disc check valve.

25. The device according to clause 18, wherein the centrifuge activatedvalve comprises a ball and spring.

26. The device according to clause 25, wherein the ball comprisesstainless steel.

27. The device according to clause 1, wherein the container furthercomprises a cap positioned at the proximal end comprising an inlet intothe cavity of the container.

28. The device according to clause 27, wherein the cap forms a fluidicseal with the internal walls of the container.

29. The device according to clause 27, wherein the cap further comprisesa vent port.

30. The device according to clause 1, further comprising a conduit thatextends from the inlet to the proximal end of the buoy.

31. The device according to clause 30, wherein the conduit is connectedto the proximal end of the buoy at a position adjacent to the inner wallof the container.

32. The device according to clause 30, wherein the conduit is releasablyattached to one or more of the inlet and the proximal end of the buoy.

33. The device according to clause 30, wherein the conduit is integratedwith one or more of the inlet or the proximal end of the buoy.

34. The device according to clause 30, wherein the conduit is releasablyattached to the inlet and integrated with the proximal end of the buoy.

35. The device according to clause 30, wherein the conduit is flexible.

36. The device according to clause 35, wherein the conduit is coiled.

37. The device according to clause 1, wherein the sealed chamberscontain a vacuum, a fluidic composition or a combination thereof.

38. The device according to clause 37, wherein one or more sealedchambers contain a vacuum.

39. The device according to clause 37, wherein one or more sealedchambers contain a fluidic composition.

40. The device according to clause 39, wherein the fluidic compositioncomprises a gaseous composition.

41. The device according to clause 40, wherein the gaseous compositioncomprises a gas selected from the group consisting of air, carbondioxide, oxygen, nitrogen, hydrogen, helium, argon, xenon or acombination thereof.

42. The device according to clause 37, wherein the fluidic compositioncomprises a liquid composition.

43. The device according to clause 42, wherein the fluidic compositioncomprises an alcohol.

44. The device according to clause 37, wherein one or more sealedchambers contain a vacuum and one or more sealed chambers contain afluidic composition.

45. The device according to clause 1, wherein the one or more sealedchambers comprises 25% or more of the total volume of the buoy.

46. The device according to clause 45, wherein the one or more sealedchambers comprises 50% or more of the total volume of the buoy.

47. The device according to clause 1, wherein the buoy comprises apolymer selected from the group consisting of polycarbonate,polycarbonate alloys, polyethylene, polypropylene,polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), ethylvinyl acetate (EVA), polyurethane, polyethersulfone, copolymers andcombinations thereof.48. The device according to clause 47, wherein the buoy comprisespolycarbonate.49. The device according to any of the preceding claims, wherein themulti-component liquid comprises a biological fluid.50. The device according to clause 49, wherein the biological fluid isselected from the group consisting of whole blood or a derivativethereof, bone marrow aspirate or a derivative thereof, stromal vascularfractions or a derivative thereof or any combination thereof.51. The device according to clause 50, wherein the biological fluid iswhole blood or a derivative thereof.52. The device according to clause 50, wherein the biological fluid isbone marrow aspirate or a derivative thereof.53. The device according to clause 50, wherein the biological fluid is astromal vascular fraction or a derivative thereof.54. The device according to clause 50, wherein the biological fluidcomprises two or more of:

whole blood or a derivative thereof;

bone marrow aspirate or a derivative thereof; and

stromal vascular fractions or a derivative thereof.

55. The device according to clause 54, wherein the biological fluidcomprises whole blood or a derivative thereof, bone marrow aspirate or aderivative thereof and stromal vascular fractions or a derivativethereof.

56. A device for separating components of a multi-component liquid, thedevice comprising:

a container comprising a buoy according to any of the preceding claims;and

a multi-component liquid present in the device.

57. The device according to clause 56, wherein the multi-componentliquid comprises a biological fluid.

58. The device according to clause 57, wherein the biological fluid isselected from the group consisting of whole blood or a derivativethereof, bone marrow aspirate or a derivative thereof, stromal vascularfractions or a derivative thereof or any combination thereof.59. The device according to clause 58, wherein the biological fluid iswhole blood or a derivative thereof.60. The device according to clause 58, wherein the biological fluid isbone marrow aspirate or a derivative thereof.61. The device according to clause 58, wherein the biological fluid is astromal vascular fraction or a derivative thereof.62. The device according to clause 58, wherein the biological fluidcomprises two or more of:

whole blood or a derivative thereof;

bone marrow aspirate or a derivative thereof; and

stromal vascular fractions or a derivative thereof.

63. The device according to clause 62, wherein the biological fluidcomprises whole blood or a derivative thereof, bone marrow aspirate or aderivative thereof and stromal vascular fractions or a derivativethereof.

64. A method comprising:

introducing a blood sample into a device comprising:

-   -   a container comprising a distal end and a proximal end; and    -   a buoy configured to be displaced along a longitudinal axis        within the container, wherein the buoy comprises one or more        sealed chambers comprising a fluidic composition;

subjecting the blood sample to a force of centrifugation to produce twoor more fractions in the blood sample;

collecting one or more components of the blood sample.

65. The method according to clause 64, wherein collecting one or morecomponents of the blood sample comprises:

removing a portion of a first fraction of the blood sample;

mixing the remaining portion of the first fraction with a secondfraction within the buoy to produce a mixture of the first fraction andthe second fraction; and

removing the mixture from the container.

66. The method according to clause 65, wherein the first fractioncomprises platelet poor plasma.

67. The method according to clause 65, wherein the second fractioncomprises platelets and white blood cells.

68. The method according to clause 65, wherein 75% by volume or more ofthe first fraction is removed from the blood sample.

69. The method according to clause 68, wherein 90% by volume or more ofthe first fraction is removed from the blood sample.

70. The method according to clause 65, wherein mixing the remainingportion of the first fraction with the second fraction comprises:

aspirating the remaining portion of the first fraction and the secondfraction into a syringe; and

reinjecting the first fraction and the second fraction within the buoyto produce a mixture of the first fraction and the second fraction.

71. The method according to clause 64, wherein the buoy comprises afrustoconical shape.

72. The method according to clause 71, wherein the buoy comprises:

a proximal end having a concave outer surface;

an orifice at the base of the concave outer surface; and

a centrifuge activated valve comprising an open position and a closedposition, wherein the valve is configured in the closed position tofluidically seal the orifice at the base of the concave outer surface.

73. The method according to clause 72, wherein the centrifuge activatedvalve is an umbrella valve.

74. The method according to clause 72, wherein the centrifuge activatedvalve is a check valve.

75. The method according to clause 72, wherein the centrifuge activatedvalve comprises a ball and spring.

76. The method according to clause 72, wherein subjecting the bloodsample to a force of centrifugation is sufficient to switch thecentrifuge activated valve to an open position.

77. The method according to clause 76, wherein subjecting the bloodsample to a force of centrifugation comprises subjecting the bloodsample to a first force of centrifugation when the centrifuge activatedvalve is in an open position and subjecting the blood sample to a secondforce of centrifugation when the centrifuge activated valve is in aclosed position.78. The method according to clause 76, wherein subjecting the bloodsample to a force of centrifugation is sufficient to collect a fractionof the blood sample on the concave outer surface of the buoy.79. The method according to clause 78, wherein subjecting the bloodsample to a force of centrifugation is sufficient to collect a fractionof the blood sample adjacent to the orifice at the base of the concaveouter surface.80. The method according to clause 64, wherein the buoy comprises:

a first orifice at the base of the concave outer surface;

a second orifice at a position distal along the longitudinal axis of thebuoy to the first orifice; and a channel that extends from the firstorifice to the second orifice;

a centrifuge activated valve comprising an open position and a closedposition, wherein the valve is configured in the closed position tofluidically seal the channel.

81. The method according to clause 80, wherein the centrifuge activatedvalve is positioned at the first orifice.

82. The method according to clause 80, wherein the centrifuge activatedvalve is positioned at the second orifice.

83. The method according to clause 80, wherein the centrifuge activatedvalve is positioned within the channel between the first orifice and thesecond orifice.

84. The method according to clause 80, wherein the centrifuge activatedvalve is an umbrella valve.

85. The method according to clause 80, wherein the centrifuge activatedvalve is a check valve.

86. The method according to clause 85, wherein the check valve comprisesa ball and spring.

87. The method according to clause 80, wherein subjecting the bloodsample to a force of centrifugation is sufficient to switch thecentrifuge activated valve to an open position.

88. The method according to clause 87, wherein subjecting the bloodsample to a force of centrifugation comprises subjecting the bloodsample to a first force of centrifugation when the centrifuge activatedvalve is in an open position and subjecting the blood sample to a secondforce of centrifugation when the centrifuge activated valve is in aclosed position.89. The method according to clause 87, wherein subjecting the bloodsample to a force of centrifugation is sufficient to collect a fractionof the blood sample in the channel.90. The method according to clause 89, wherein subjecting the bloodsample to a force of centrifugation is sufficient to collect a fractionof the blood sample in the channel adjacent to the first orifice.91. The method according to clause 89, wherein subjecting the bloodsample to a force of centrifugation is sufficient to collect a fractionof the blood sample in the channel adjacent to the second orifice.92. The method according to clause 89, wherein subjecting the bloodsample to a force of centrifugation is sufficient to collect a fractionof the blood sample on the concave outer surface of the buoy.93. The method according to clause 87, wherein subjecting the bloodsample to a force of centrifugation is sufficient to collect a fractionof the blood sample on the concave outer surface of the buoy adjacent tothe first orifice.94. The method according to clause 64, wherein collecting one or morecomponents of the biological sample comprises:

positioning the device at a first angle with respect to an axisorthogonal to the ground;

removing a portion of a first fraction of the biological sample;

rotating the device by a second angle along the longitudinal axis of thedevice;

aspirating the remaining portion of the first fraction of the biologicalsample through the conduit;

mixing the remaining portion of the first fraction with a secondfraction within the buoy to produce a mixture of the first fraction andsecond fraction; and

removing the mixture from the container.

95. The method according to clause 64, wherein collecting one or morecomponents of the biological sample comprises:

positioning the device at a first angular position with respect to anaxis orthogonal to the ground;

removing a portion of a first fraction of the biological sample;

tilting the device to a second angular position with respect to the axisorthogonal to the ground;

aspirating the remaining portion of the first fraction of the biologicalsample;

mixing the remaining portion of the first fraction with a secondfraction within the buoy to produce a mixture of the first fraction andthe second fraction; and

removing the mixture from the container.

96. The method according to clause 95, wherein the second angularposition is from 5° to 20° with respect to the first angular position ofthe device.

97. The method according to clause 95, wherein the second angularposition is 10° with respect to the first angular position.

98. The method according to clause 64, wherein the container comprises:

a cap positioned at the proximal end comprising an inlet into the cavityof the container; and

a conduit that connects the inlet to the proximal end of the buoy.

99. The method according to clause 98, wherein the conduit is connectedto the proximal end of the buoy at a position adjacent to the inner wallof the container.

100. The method according to clause 99, wherein collecting one or morecomponents of the blood sample comprises:

positioning the container at a first angle with respect to an axisorthogonal to the ground;

removing a portion of a first fraction of the blood sample through theconduit;

rotating the container by a second angle along the longitudinal axis ofthe container;

aspirating the remaining portion of the first fraction of the bloodsample through the conduit;

mixing the remaining portion of the first fraction with a secondfraction within the buoy to produce a mixture of the first fraction andsecond fraction; and

removing the mixture from the container.

101. The method according to clause 99, wherein collecting one or morecomponents of the blood sample comprises:

positioning the device at a first angular position with respect to anaxis orthogonal to the ground;

removing a portion of a first fraction of the blood sample through theconduit;

tilting the device to a second angular position with respect to the axisorthogonal to the ground;

aspirating the remaining portion of the first fraction of the bloodsample through the conduit;

mixing the remaining portion of the first fraction with a secondfraction within the buoy to produce a mixture of the first fraction andthe second fraction; and

removing the mixture from the container.

102. The method according to clause 101, wherein the second angularposition is from 5° to 20° with respect to the first angular position ofthe device.

103. The method according to clause 101, wherein the second angularposition is 10° with respect to the first angular position.

104. The method according to any of clauses 100-101, wherein the firstfraction comprises platelet poor plasma.

105. The method according to any of clauses 100-101, wherein the secondfraction comprises platelets and white blood cells.

106. The method according to any of clauses 100-101, wherein 75% byvolume or more of the first fraction is removed from the blood sample.

107. The method according to any of clauses 100-101, wherein 90% byvolume or more of the first fraction is removed from the blood sample.

108. A method comprising: introducing a biological sample into a devicecomprising:

a container comprising a distal end and a proximal end; and

a buoy configured to be displaced along a longitudinal axis within thecontainer, wherein the buoy comprises one or more sealed chamberscomprising a fluidic composition;

subjecting the biological sample to a force of centrifugation to producetwo or more fractions in the blood sample;

collecting one or more components of the biological sample.

109. The method according to clause 108, wherein collecting one or morecomponents of the blood biological comprises:

removing a portion of a first fraction of the biological sample;

mixing the remaining portion of the first fraction with a secondfraction within the buoy to produce a mixture of the first fraction andthe second fraction; and

removing the mixture from the container.

110. The method according to clause 109, wherein the first fractioncomprises platelet poor plasma.

111. The method according to clause 109, wherein the second fractioncomprises platelets and white blood cells.

112. The method according to clause 109, wherein 75% by volume or moreof the first fraction is removed from the biological sample.

113. The method according to clause 109, wherein 90% by volume or moreof the first fraction is removed from the biological sample.

114. The method according to clause 109, wherein mixing the remainingportion of the first fraction with the second fraction comprises:

aspirating the remaining portion of the first fraction and the secondfraction into a syringe; and

reinjecting the first fraction and the second fraction within the buoyto produce a mixture of the first fraction and the second fraction.

115. The method according to clause 108, wherein the buoy comprises afrustoconical shape.

116. The method according to clause 115, wherein the buoy comprises:

a proximal end having a concave outer surface;

an orifice at the base of the concave outer surface; and

a centrifuge activated valve comprising an open position and a closedposition, wherein the valve is configured in the closed position tofluidically seal the orifice at the base of the concave outer surface.

117. The method according to clause 116, wherein the centrifugeactivated valve is an umbrella valve.

118. The method according to clause 116, wherein the centrifugeactivated valve is a check valve.

119. The method according to clause 116, wherein the centrifugeactivated valve comprises a ball and spring.

120. The method according to clause 116, wherein subjecting thebiological sample to a force of centrifugation is sufficient to switchthe centrifuge activated valve to an open position.

121. The method according to clause 120, wherein subjecting thebiological sample to a force of centrifugation comprises subjecting thebiological sample to a first force of centrifugation when the centrifugeactivated valve is in an open position and subjecting the biologicalsample to a second force of centrifugation when the centrifuge activatedvalve is in a closed position.122. The method according to clause 116, wherein subjecting thebiological sample to a force of centrifugation is sufficient to collecta fraction of the biological sample on the concave outer surface of thebuoy.123. The method according to clause 122, wherein subjecting thebiological sample to a force of centrifugation is sufficient to collecta fraction of the biological sample adjacent to the orifice at the baseof the concave outer surface.124. The method according to clause 116, wherein the buoy comprises:

a first orifice at the base of the concave outer surface;

a second orifice at a position distal along the longitudinal axis of thebuoy to the first orifice; and a channel that extends from the firstorifice to the second orifice;

a centrifuge activated valve comprising an open position and a closedposition, wherein the valve is configured in the closed position tofluidically seal the channel.

125. The method according to clause 124, wherein the centrifugeactivated valve is positioned at the first orifice.

126. The method according to clause 124, wherein the centrifugeactivated valve is positioned at the second orifice.

127. The method according to clause 124, wherein the centrifugeactivated valve is positioned within the channel between the firstorifice and the second orifice.

128. The method according to clause 124, wherein the centrifugeactivated valve is an umbrella valve.

129. The method according to clause 124, wherein the centrifugeactivated valve is a check valve.

130. The method according to clause 129, wherein the check valvecomprises a ball and spring.

131. The method according to clause 124, wherein subjecting thebiological sample to a force of centrifugation is sufficient to switchthe centrifuge activated valve to an open position.

132. The method according to clause 131, wherein subjecting thebiological sample to a force of centrifugation comprises subjecting thebiological sample to a first force of centrifugation when the centrifugeactivated valve is in an open position and subjecting the biologicalsample to a second force of centrifugation when the centrifuge activatedvalve is in a closed position.133. The method according to clause 131, wherein subjecting thebiological sample to a force of centrifugation is sufficient to collecta fraction of the biological sample in the channel.134. The method according to clause 133, wherein subjecting thebiological sample to a force of centrifugation is sufficient to collecta fraction of the biological sample in the channel adjacent to the firstorifice.135. The method according to clause 133, wherein subjecting thebiological sample to a force of centrifugation is sufficient to collecta fraction of the biological sample in the channel adjacent to thesecond orifice.136. The method according to clause 133, wherein subjecting thebiological sample to a force of centrifugation is sufficient to collecta fraction of the biological sample on the concave outer surface of thebuoy.137. The method according to clause 133, wherein subjecting thebiological sample to a force of centrifugation is sufficient to collecta fraction of the biological sample on the concave outer surface of thebuoy adjacent to the first orifice.138. The method according to clause 116, wherein collecting one or morecomponents of the biological sample comprises:

positioning the device at a first angle with respect to an axisorthogonal to the ground;

removing a portion of a first fraction of the biological sample;

rotating the device by a second angle along the longitudinal axis of thedevice;

aspirating the remaining portion of the first fraction of the biologicalsample through the conduit;

mixing the remaining portion of the first fraction with a secondfraction within the buoy to produce a mixture of the first fraction andsecond fraction; and

removing the mixture from the container.

139. The method according to clause 116, wherein collecting one or morecomponents of the biological sample comprises:

positioning the device at a first angular position with respect to anaxis orthogonal to the ground;

removing a portion of a first fraction of the biological sample;

tilting the device to a second angular position with respect to the axisorthogonal to the ground;

aspirating the remaining portion of the first fraction of the biologicalsample;

mixing the remaining portion of the first fraction with a secondfraction within the buoy to produce a mixture of the first fraction andthe second fraction; and

removing the mixture from the container.

140. The method according to clause 139, wherein the second angularposition is from 5° to 20° with respect to the first angular position ofthe device.

141. The method according to clause 139, wherein the second angularposition is 10° with respect to the first angular position.

142. The method according to clause 116, wherein the containercomprises:

a cap positioned at the proximal end comprising an inlet into the cavityof the container; and a conduit that connects the inlet to the proximalend of the buoy.

143. The method according to clause 142, wherein the conduit isconnected to the proximal end of the buoy at a position adjacent to theinner wall of the container.

144. The method according to clause 143, wherein collecting one or morecomponents of the biological sample comprises:

positioning the container at a first angle with respect to an axisorthogonal to the ground;

removing a portion of a first fraction of the biological sample throughthe conduit;

rotating the container by a second angle along the longitudinal axis ofthe container;

aspirating the remaining portion of the first fraction of the biologicalsample through the conduit;

mixing the remaining portion of the first fraction with a secondfraction within the buoy to produce a mixture of the first fraction andsecond fraction; and

removing the mixture from the container.

145. The method according to clause 143, wherein collecting one or morecomponents of the biological sample comprises:

positioning the device at a first angular position with respect to anaxis orthogonal to the ground;

removing a portion of a first fraction of the biological sample throughthe conduit;

tilting the device to a second angular position with respect to the axisorthogonal to the ground;

aspirating the remaining portion of the first fraction of the biologicalsample through the conduit;

mixing the remaining portion of the first fraction with a secondfraction within the buoy to produce a mixture of the first fraction andthe second fraction; and

removing the mixture from the container.

146. The method according to clause 145, wherein the second angularposition is from 5° to 20° with respect to the first angular position ofthe device.

147. The method according to clause 145, wherein the second angularposition is 10° with respect to the first angular position.

148. The method according to clause 147, wherein the first fractioncomprises platelet poor plasma.

149. The method according to clause 147, wherein the second fractioncomprises platelets and white blood cells.

150. The method according to clause 147, wherein 75% by volume or moreof the first fraction is removed from the biological sample.

151. The method according to clause 147, wherein 90% by volume or moreof the first fraction is removed from the biological sample.

152. The method according to any of clauses 116-151, wherein thebiological sample is selected from the group consisting of whole blood,bone marrow aspirate, stromal vascular fraction and a combinationthereof.

153. The method according to any of clauses 116-151, wherein thebiological sample is whole blood.

154. The method according to any of clauses 116-151, wherein thebiological sample is bone marrow aspirate.

155. The method according to any of clauses 116-151, wherein thebiological sample is stromal vascular fraction.

156. A method comprising:

introducing a multicomponent liquid sample into a device comprising:

-   -   a container comprising a distal end and a proximal end; and    -   a buoy configured to be displaced along a longitudinal axis        within the container, wherein the buoy comprises one or more        sealed chambers comprising a fluidic composition;

subjecting the multicomponent liquid sample to a force of centrifugationto produce two or more fractions in the multicomponent liquid sample;

collecting one or more components from the separated fractions.

157. The method according to clause 156, wherein the buoy comprises afrustoconical shape.

158. The method according to clause 157, wherein the buoy comprises:

a proximal end having a concave outer surface;

an orifice at the base of the concave outer surface; and

a centrifuge activated valve comprising an open position and a closedposition, wherein the valve is configured in the closed position tofluidically seal the orifice at the base of the concave outer surface.

159. The method according to clause 158, wherein the centrifugeactivated valve comprises a ball and spring.

160. The method according to clause 158, wherein subjecting themulticomponent liquid sample to a force of centrifugation is sufficientto switch the centrifuge activated valve to an open position.

161. The method according to clause 158, wherein subjecting themulticomponent liquid sample to a force of centrifugation is sufficientto collect a fraction on the concave outer surface of the buoy.

162. The method according to clause 161, wherein subjecting themulticomponent liquid sample to a force of centrifugation is sufficientto collect a fraction adjacent to the orifice at the base of the concaveouter surface.

163. The method according to clause 158, wherein the buoy comprises:

a first orifice at the base of the concave outer surface;

a second orifice at a position distal along the longitudinal axis of thebuoy to the first orifice; and

a channel that extends from the first orifice to the second orifice;

a centrifuge activated valve comprising an open position and a closedposition, wherein the valve is configured in the closed position tofluidically seal the channel.

164. The method according to clause 163, wherein the centrifugeactivated valve is positioned at the first orifice.

165. The method according to clause 163, wherein the centrifugeactivated valve is positioned at the second orifice.

166. The method according to clause 163, wherein the centrifugeactivated valve is positioned within the channel between the firstorifice and the second orifice.

167. The method according to clause 163, wherein the centrifugeactivated valve is an umbrella valve.

168. The method according to clause 163, wherein the centrifugeactivated valve is a check valve.

169. The method according to clause 168, wherein the check valvecomprises a ball and spring.

170. The method according to clause 163, wherein subjecting themulticomponent liquid sample to a force of centrifugation is sufficientto switch the centrifuge activated valve to an open position.

171. The method according to clause 170, wherein subjecting themulticomponent liquid sample to a force of centrifugation comprisessubjecting the blood sample to a first force of centrifugation when thecentrifuge activated valve is in an open position and subjecting themulticomponent liquid sample to a second force of centrifugation whenthe centrifuge activated valve is in a closed position.172. The method according to clause 170, wherein subjecting themulticomponent liquid sample to a force of centrifugation is sufficientto collect a fraction in the channel.173. The method according to clause 172, wherein subjecting themulticomponent liquid sample to a force of centrifugation is sufficientto collect a fraction in the channel adjacent to the first orifice.174. The method according to clause 172, wherein subjecting themulticomponent liquid sample to a force of centrifugation is sufficientto collect a fraction in the channel adjacent to the second orifice.175. The method according to clause 174, wherein subjecting themulticomponent liquid sample to a force of centrifugation is sufficientto collect a fraction on the concave outer surface of the buoy.176. The method according to clause 174, wherein subjecting themulticomponent liquid sample to a force of centrifugation is sufficientto collect a fraction on the concave outer surface of the buoy adjacentto the first orifice.177. The method according to clause 156, wherein collecting one or morecomponents of the multicomponent sample comprises:

positioning the container at a first angle with respect to an axisorthogonal to the ground;

removing a portion of a first fraction;

positioning the container at a second angle with respect to an axisorthogonal to the ground;

aspirating the remaining portion of the first fraction;

mixing the remaining portion of the first fraction with a secondfraction within the buoy to produce a mixture of the first fraction andsecond fraction; and

removing the mixture from the container.

178. The method according to clause 156, wherein collecting one or morecomponents of the multicomponent sample comprises:

positioning the device at a first angular position with respect to anaxis orthogonal to the ground;

removing a portion of a first fraction of the sample;

tilting the device to a second angular position with respect to the axisorthogonal to the ground;

aspirating the remaining portion of the first fraction of the sample;

mixing the remaining portion of the first fraction with a secondfraction within the buoy to produce a mixture of the first fraction andthe second fraction; and

removing the mixture from the container.

179. The method according to clause 178, wherein the second angularposition is from 5° to 20° with respect to the first angular position ofthe device.

180. The method according to clause 178, wherein the second angularposition is 10° with respect to the first angular position.

181. The method according to clause 156, wherein the containercomprises:

a cap positioned at the proximal end comprising an inlet into the cavityof the container; and

a conduit that connects the inlet to the proximal end of the buoy.

182. The method according to clause 181, wherein the conduit isconnected to the proximal end of the buoy at a position adjacent to theinner wall of the container.

183. The method according to clause 181, wherein the cap comprises asingle inlet into the cavity of the container.

184. The method according to clause 181, wherein the conduit furthercomprises a stream modulator.

185. The method according to clause 184, wherein the stream modulator isconfigured to adjust a rate of fluid output from the conduit.

186. The method according to clause 184, wherein the stream modulator isconfigured to adjust a shape fluid output from the conduit.

187. The method according to clause 184, wherein the stream modulator isconfigured to adjust the cross-sectional dimensions of fluid output fromthe conduit.

188. The method according to clause 156, wherein collecting one or morecomponents of the multicomponent sample comprises:

positioning the container at a first angle with respect to an axisorthogonal to the ground;

removing a portion of a first fraction through the conduit;

positioning the container at a second angle with respect to an axisorthogonal to the ground;

aspirating the remaining portion of the first fraction through theconduit;

mixing the remaining portion of the first fraction with a secondfraction within the buoy to produce a mixture of the first fraction andsecond fraction; and

removing the mixture from the container.

189. The method according to clause 156, wherein collecting one or morecomponents of the multicomponent sample comprises:

positioning the device at a first angular position with respect to anaxis orthogonal to the ground;

removing a portion of a first fraction through the conduit;

tilting the device to a second angular position with respect to the axisorthogonal to the ground;

aspirating the remaining portion of the first fraction through theconduit;

mixing the remaining portion of the first fraction with a secondfraction within the buoy to produce a mixture of the first fraction andthe second fraction; and

removing the mixture from the container.

190. The method according to clause 189, wherein the second angularposition is from 5° to 20° with respect to the first angular position ofthe device.

191. The method according to clause 189, wherein the second angularposition is 10° with respect to the first angular position.

192. A system comprising:

a device for separating components of a multi-component liquidcomprising:

a container comprising a distal end and a proximal end, wherein thecontainer comprises a cap positioned at the proximal end comprising aninlet into the cavity of the container; and

a buoy configured to be displaced along a longitudinal axis within thecontainer, wherein the buoy comprises one or more sealed chambers; and

a support for positioning the container at an angle with respect to anaxis orthogonal to the ground.

193. The system according to clause 192, wherein the container furthercomprises a cap positioned at the proximal end comprising an inlet intothe cavity of the container.

194. The system according to clause 193, wherein the cap comprises asingle inlet into the cavity of the container.

195. The system according to clause 193, wherein the cap forms a fluidicseal with the internal walls of the container.

196. The system according to clause 193, wherein the cap furthercomprises a vent port.

197. The system according to clause 192, further comprising a conduitthat extends from the inlet to the proximal end of the buoy.

198. The system according to clause 197, wherein the conduit isconnected to the proximal end of the buoy at a position adjacent to theinner wall of the container.

199. The system according to clause 197, wherein the conduit isreleasably attached to one or more of the inlet and the proximal end ofthe buoy.

200. The system according to clause 197, wherein the conduit isintegrated with one or more of the inlet or the proximal end of thebuoy.

201. The system according to clause 197, wherein the conduit isreleasably attached to the inlet and integrated with the proximal end ofthe buoy.

202. The system according to clause 197, wherein the conduit isflexible.

203. The system according to clause 202, wherein the conduit is coiled.

204. The system according to clause 197, wherein the conduit furthercomprises a stream modulator.

205. The system according to clause 204, wherein the stream modulator isconfigured to adjust a rate of fluid output from the conduit.

206. The system according to clause 204, wherein the stream modulator isconfigured to adjust a shape fluid output from the conduit.

207. The system according to clause 204, wherein the stream modulator isconfigured to adjust the cross-sectional dimensions of fluid output fromthe conduit.

208. The system according to clause 192, wherein the support isconfigured to position the container at an angle that is 10 degrees ormore with respect to an axis orthogonal to the ground.

209. The system according to clause 192, wherein the support isadjustable to position the container at an angle that is from 10 degreesto 90 degrees with respect to an axis orthogonal to the ground.

210. The system according to clause 209, wherein the support furthercomprises an actuator to adjust the position of the container whenpositioned in the support.

211. The system according to clause 210, wherein the actuator ismechanical.

212. The system according to clause 210, wherein the actuator ismotor-powered.

213. The system according to clause 192, wherein the support isconfigured for positioning the container at a second angle.

214. The system according to clause 213, wherein the support isconfigured for rotating the container an angle of 45 degrees or morealong a longitudinal axis of the container.

215. The system according to clause 213, wherein the support isconfigured to rotate the container an angle of 180 degrees or more alonga longitudinal axis of the container.

215. The system according to clause 192, wherein the buoy comprises afrustoconical shaped component.

216. The system according to clause 215, wherein the buoy comprises acylindrical proximal portion and frustoconical distal portion.

217. The system according to clause 215, wherein the proximal end of thebuoy comprises a concave outer surface.

218. The system according to clause 192, wherein the sealed chamberscontain a vacuum, a fluidic composition or a combination thereof 219.The system according to clause 192, wherein one or more sealed chamberscontain a vacuum.

220. The system according to clause 192, wherein one or more sealedchambers contain a fluidic composition.

221. The system according to clause 192, wherein the fluidic compositioncomprises a gaseous composition.

222. The system according to clause 192, wherein the gaseous compositioncomprises a gas selected from the group consisting of air, carbondioxide, oxygen, nitrogen, hydrogen, helium, argon, xenon or acombination thereof 223. The system according to clause 192, wherein thefluidic composition comprises a liquid composition.224. The system according to clause 192, wherein the fluidic compositioncomprises an alcohol.225. The system according to clause 192, wherein one or more sealedchambers contain a vacuum and one or more sealed chambers contain afluidic composition.226. The system according to clause 192, wherein the buoy comprises:

an orifice at the base of the concave outer surface; and

a centrifuge activated valve comprising an open position and a closedposition, wherein the valve is configured in the closed position tofluidically seal the orifice at the base of the concave outer surface.

227. The system according to clause 226, wherein the centrifugeactivated valve is an umbrella valve.

228. The system according to clause 226, wherein the centrifugeactivated valve is a check valve.

229. The system according to clause 228, wherein the check valve isselected from the group consisting of a ball check valve, a diaphragmcheck valve, a lift check valve and a tilted disc check valve.

230. The system according to clause 226, wherein the centrifugeactivated valve comprises a ball and spring.

231. The system according to clause 230, wherein the ball comprisesstainless steel.

232. The system according to clause 192, wherein the buoy comprises:

a first orifice at the base of the concave outer surface;

a second orifice at a position distal along the longitudinal axis of thebuoy to the first orifice; and

a channel that extends from the first orifice to the second orifice;

a centrifuge activated valve comprising an open position and a closedposition, wherein the valve is configured in the closed position tofluidically seal the channel.

233. The system according to clause 232, wherein the centrifugeactivated valve is positioned at the first orifice.

234. The system according to clause 232, wherein the centrifugeactivated valve is positioned at the second orifice.

235. The system according to clause 232, wherein the centrifugeactivated valve is positioned within the channel between the firstorifice and the second orifice.

236. The system according to clause 235, wherein the centrifugeactivated valve is an umbrella valve.

237. The system according to clause 232, wherein the centrifugeactivated valve is a check valve.

238. The system according to clause 237, wherein the check valve isselected from the group consisting of a ball check valve, a diaphragmcheck valve, a lift check valve and a tilted disc check valve.

239. The system according to clause 237, wherein the centrifugeactivated valve comprises a ball and spring.

240. The system according to clause 239, wherein the ball comprisesstainless steel.

241. The system according to clause 192, wherein the gaseous compositioncomprises a compound selected from the group consisting of air, carbondioxide, oxygen, nitrogen, hydrogen, helium, argon, xenon or acombination thereof.

242. The system according to clause 241, wherein the gaseous compositioncomprises air.

243. The system according to clause 192, wherein the one or more sealedchambers comprises 25% or more of the total volume of the buoy.

244. The system according to clause 243, wherein the one or more sealedchambers comprises 50% or more of the total volume of the buoy.

245. The system according to clause 192, wherein the buoy comprises apolymer selected from the group consisting of polycarbonate,polycarbonate alloys, polyethylene, polypropylene,polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), ethylvinyl acetate (EVA), polyurethane, polyethersulfone, copolymers andcombinations thereof.246. The system according to clause 245, wherein the buoy comprisespolycarbonate.247. The system according to any of the preceding clause, wherein themulti-component liquid is a biological sample or a derivative thereof.248. The method according to clause 247, wherein the biological sampleis selected from the group consisting of whole blood, bone marrowaspirate, stromal vascular fraction and a combination thereof.249. The method according to any of the preceding clauses, wherein thebiological sample is whole blood.250. The method according to any of the preceding clauses, wherein thebiological sample is bone marrow aspirate.251. The method according to any of the preceding clauses, wherein thebiological sample is stromal vascular fraction.252. A system comprising:

a centrifuge;

a device for separating components of blood, the device comprising:

-   -   a container comprising a distal end and a proximal end; and    -   a buoy configured to be displaced along a longitudinal axis        within the container, wherein the buoy comprises one or more        sealed chambers.        253. The system according to clause 252, wherein the centrifuge        is a fixed angle centrifuge.        254. The system according to clause 252, wherein the centrifuge        is a swinging bucket centrifuge.        255. The system according to clause 252, wherein the proximal        end of the buoy comprises a concave outer surface.        256. The system according to clause 255, wherein the buoy        comprises:

an orifice at the base of the concave outer surface; and

a centrifuge activated valve comprising an open position and a closedposition, wherein the valve is configured in the closed position tofluidically seal the orifice at the base of the concave outer surface.

257. The system according to clause 256, wherein the centrifugeactivated valve is an umbrella valve.

258. The system according to clause 256, wherein the centrifugeactivated valve is a check valve.

259. The system according to clause 257, wherein the check valve isselected from the group consisting of a ball check valve, a diaphragmcheck valve, a lift check valve and a tilted disc check valve.

260. The system according to clause 259, wherein the centrifugeactivated valve comprises a ball and spring.

261. The system according to clause 260, wherein the ball comprisesstainless steel.

262. The system according to clause 252, wherein the buoy comprises:

a first orifice at the base of the concave outer surface;

a second orifice at a position distal along the longitudinal axis of thebuoy to the first orifice; and

a channel that extends from the first orifice to the second orifice;

a centrifuge activated valve comprising an open position and a closedposition, wherein the valve is configured in the closed position tofluidically seal the channel.

263. The system according to clause 262, wherein the centrifugeactivated valve is positioned at the first orifice.

264. The system according to clause 262, wherein the centrifugeactivated valve is positioned at the second orifice.

265. The system according to clause 262, wherein the centrifugeactivated valve is positioned within the channel between the firstorifice and the second orifice.

266. The system according to clause 262, wherein the centrifugeactivated valve is an umbrella valve.

267. The system according to clause 262, wherein the centrifugeactivated valve is a check valve.

268. The system according to clause 267, wherein the check valve isselected from the group consisting of a ball check valve, a diaphragmcheck valve, a lift check valve and a tilted disc check valve.

269. The system according to clause 267, wherein the centrifugeactivated valve comprises a ball and spring.

270. The system according to clause 269, wherein the ball comprisesstainless steel.

271. The system according to clause 252, wherein the container furthercomprises a cap positioned at the proximal end comprising an inlet intothe cavity of the container.

272. The system according to clause 271, wherein the cap comprises asingle inlet into the cavity of the container.

273. The system according to clause 271, wherein the cap forms a fluidicseal with the internal walls of the container.

274. The system according to clause 271, wherein the cap furthercomprises a vent port.

275. The system according to clause 271, further comprising a conduitthat extends from the inlet to the proximal end of the buoy.

276. The system according to clause 275, wherein the conduit isconnected to the proximal end of the buoy at a position adjacent to theinner wall of the container.

277. The system according to clause 276, wherein the conduit isreleasably attached to one or more of the inlet and the proximal end ofthe buoy.

278. The system according to clause 276, wherein the conduit isintegrated with one or more of the inlet or the proximal end of thebuoy.

279. The system according to clause 276, wherein the conduit isreleasably attached to the inlet and integrated with the proximal end ofthe buoy.

280. The system according to claim 276, wherein the conduit is flexible.

281. The system according to claim 276, wherein the conduit is coiled.

282. The system according to clause 275, wherein the conduit furthercomprises a stream modulator.

283. The system according to clause 282, wherein the stream modulator isconfigured to adjust a rate of fluid output from the conduit.

284. The system according to clause 282, wherein the stream modulator isconfigured to adjust a shape fluid output from the conduit.

285. The system according to clause 282, wherein the stream modulator isconfigured to adjust the cross-sectional dimensions of fluid output fromthe conduit.

286. The system according to clause 252, wherein the sealed chamberscontain a vacuum, a fluidic composition or a combination thereof.

287. The system according to clause 286, wherein one or more sealedchambers contain a vacuum.

288. The system according to clause 286, wherein one or more sealedchambers contain a fluidic composition.

289. The system according to clause 288, wherein the fluidic compositioncomprises a gaseous composition.

290. The system according to clause 289, wherein the gaseous compositioncomprises a compound selected from the group consisting of air, carbondioxide, oxygen, nitrogen, hydrogen, helium, argon, xenon or acombination thereof.

291. The system according to clause 290, wherein the gaseous compositioncomprises air.

292. The system according to clause 288, wherein the fluidic compositioncomprises a liquid composition.

293. The system according to clause 292, wherein the fluidic compositioncomprises an alcohol.

294. The system according to clause 252, wherein one or more sealedchambers contain a vacuum and one or more sealed chambers contain afluidic composition.

295. The system according to clause 252, wherein the one or more sealedchambers comprises 25% or more of the total volume of the buoy.

296. The system according to clause 252, wherein the one or more sealedchambers comprises 50% or more of the total volume of the buoy.

297. The system according to clause 252, wherein the buoy comprises apolymer selected from the group consisting of polycarbonate,polycarbonate alloys, polyethylene, polypropylene,polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), ethylvinyl acetate (EVA), polyurethane, polyethersulfone, copolymers andcombinations thereof 160. The system according to clause 159, whereinthe buoy comprises polycarbonate.298. A kit comprising:

a device for separating components of a multi-component liquid, thedevice comprising: a container comprising a distal end and a proximalend; and a buoy configured to be displaced along a longitudinal axiswithin the container, wherein the buoy comprises one or more sealedchambers comprising a fluidic composition; and

a container housing the device.

299. The kit according to clause 298, wherein the container comprises apouch.

300. The kit according to clause 298, further comprising a syringe.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this disclosure that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention being withoutlimitation to such specifically recited examples and conditions.Moreover, all statements herein reciting principles, aspects, andembodiments of the invention as well as specific examples thereof, areintended to encompass both structural and functional equivalentsthereof. Additionally, it is intended that such equivalents include bothcurrently known equivalents and equivalents developed in the future,i.e., any elements developed that perform the same function, regardlessof structure. The scope of the present invention, therefore, is notintended to be limited to the exemplary embodiments shown and describedherein. Rather, the scope and spirit of the present invention isembodied by the appended claims.

What is claimed is:
 1. A method comprising: introducing a biologicalsample into a device for separating components of a multi-componentliquid, the device comprising: a container comprising a distal end and aproximal end; and a buoy having a distal end and a proximal end and isconfigured to be displaced along a longitudinal axis within thecontainer, wherein the buoy comprises: one or more chambers containing avacuum or a fluid and sealed from fluidic communication with an outsideenvironment of the buoy; a concave first outer surface at the proximalend of the buoy, the first surface comprising a first orifice at thebase of the concave outer surface, wherein the buoy does not comprise avalve at the first orifice; a second outer surface at the distal end ofthe buoy comprising a second orifice at a position distal along thelongitudinal axis of the buoy to the first orifice, the first orifice influid communication with the second orifice through a channel extendingfrom the first orifice to the second orifice; and a centrifuge activatedball and spring valve at the second orifice comprising an open positionand a closed position; wherein the valve is configured to be in theclosed position to fluidically seal the second orifice withoutcentrifugation, wherein the valve is configured to continuously be inthe open position during centrifugation in response to an applied forceof centrifugation, and wherein the ball of the valve is configured tocompress in response to the applied force of centrifugation in the openposition; subjecting the biological sample to a force of centrifugationto produce two or more fractions in the biological sample; andcollecting one or more fractions or portions of fractions of thebiological sample.
 2. The method according to claim 1, whereincollecting one or more components of the biological sample comprises:removing a portion of a first fraction of the biological sample; mixingthe remaining portion of the first fraction with a second fractionwithin the buoy to produce a mixture of the first fraction and thesecond fraction; and removing the mixture from the container.
 3. Themethod according to claim 1, wherein collecting one or more componentsof the biological sample comprises: positioning the device at a firstangle with respect to an axis orthogonal to the ground; removing aportion of a first fraction of the biological sample; rotating thedevice by a second angle along the longitudinal axis of the container;aspirating the remaining portion of the first fraction of the biologicalsample; mixing the remaining portion of the first fraction with a secondfraction within the buoy to produce a mixture of the first fraction andsecond fraction; and removing the mixture from the container.
 4. Themethod according to claim 3, wherein the first angle is 10 degrees ormore with respect to an axis orthogonal to the ground.
 5. The methodaccording to claim 3, wherein the first angle is 10 degrees to 90degrees with respect to an axis orthogonal to the ground.
 6. The methodaccording to claim 3, wherein the second angle is 45 degrees or morealong the longitudinal axis of the container.
 7. The method according toclaim 3, wherein the second angle is 180 degrees or more along thelongitudinal axis of the container.
 8. The method according to claim 3,wherein the device is positioned at the first angle and the second angleusing a support configured for positioning the device at the first angleand second angle.
 9. The method according to claim 1, wherein collectingone or more components of the biological sample comprises: positioningthe device at a first angular position with respect to an axisorthogonal to the ground; removing a portion of a first fraction of thebiological sample; tilting the device to a second angular position withrespect to the axis orthogonal to the ground; aspirating the remainingportion of the first fraction of the biological sample; mixing theremaining portion of the first fraction with a second fraction withinthe buoy to produce a mixture of the first fraction and the secondfraction; and removing the mixture from the container.
 10. The methodaccording to claim 1, wherein the biological fluid is selected from thegroup consisting of whole blood or a derivative thereof, bone marrowaspirate or a derivative thereof, stromal vascular fractions or aderivative thereof or any combination thereof.
 11. The method accordingto claim 1, wherein the biological fluid is whole blood or a derivativethereof.
 12. The method according to claim 11, wherein a first fractioncomprises platelet poor plasma and a second fraction comprises plateletsand white blood cells.
 13. The method according to claim 11, wherein 75%to 95% by volume of a first fraction is removed from the whole bloodsample.
 14. The method according to claim 11, wherein subjecting thewhole blood sample to a force of centrifugation is sufficient to switchthe centrifuge activated valve to an open position.
 15. The methodaccording to claim 14, wherein subjecting the blood sample to a force ofcentrifugation comprises subjecting the blood sample to a first force ofcentrifugation when the centrifuge activated valve is in an openposition and subjecting the blood sample to a second force ofcentrifugation when the centrifuge activated valve is in a closedposition.
 16. The method according to claim 14, wherein subjecting theblood sample to a force of centrifugation is sufficient to collect afraction of the blood sample in the channel.
 17. The method according toclaim 16, wherein subjecting the blood sample to a force ofcentrifugation is sufficient to collect a fraction of the blood samplein the channel adjacent to the first orifice.
 18. The method accordingto claim 16, wherein subjecting the blood sample to a force ofcentrifugation is sufficient to collect a fraction of the blood samplein the channel adjacent to the second orifice.
 19. The method accordingto claim 16, wherein subjecting the blood sample to a force ofcentrifugation is sufficient to collect a fraction of the blood sampleon the concave outer surface of the buoy.
 20. A system comprising: (a) adevice for separating components of a multi-component liquid, the devicecomprising: a container comprising a distal end and a proximal end; anda buoy having a distal end and a proximal end and is configured to bedisplaced along a longitudinal axis within the container, wherein thebuoy comprises: one or more chambers containing a vacuum or a fluid andsealed from fluidic communication with an outside environment of thebuoy; a concave first outer surface at the proximal end of the buoy, thefirst surface comprising a first orifice at the base of the concaveouter surface, wherein the buoy does not comprise a valve at the firstorifice; a second outer surface at the distal end of the buoy comprisinga second orifice at a position distal along the longitudinal axis of thebuoy to the first orifice, the first orifice in fluid communication withthe second orifice through a channel extending from the first orifice tothe second orifice; and a centrifuge activated ball and spring valve atthe second orifice comprising an open position and a closed position;wherein the valve is configured to be in the closed position tofluidically seal the second orifice without centrifugation, wherein thevalve is configured to continuously be in the open position duringcentrifugation in response to a force of centrifugation, and wherein theball of the valve is configured to compress in response to the appliedforce of centrifugation in the open position; (b) a support forpositioning the container at an angle with respect to an axis orthogonalto the ground; or (c) a centrifuge.
 21. The system according to claim20, wherein the system comprises the device for separating components ofthe multi-component liquid and the support.
 22. The system according toclaim 21, wherein the support is adjustable to position the container atan angle that is from 10 degrees to 90 degrees with respect to an axisorthogonal to the ground.
 23. The system according to claim 22, whereinthe support further comprises an actuator to adjust the position of thecontainer when positioned in the support.
 24. The system according toclaim 22, wherein the system comprises the device for separatingcomponents of the multi-component liquid and the centrifuge.